Methods, compositions and kits pertaining to analyte determination

ABSTRACT

This invention pertains to methods, kits and/or compositions for the determination of analytes by mass analysis using unique labeling reagents or sets of unique labeling reagents. The labeling reagents can be isomeric or isobaric and can be used to produce mixtures suitable for multiplex analysis of the labeled analytes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/765,267 filed on Jan. 27, 2004, incorporated herein by reference,which application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/443,612, filed on Jan. 30, 2003, incorporatedherein by reference.

FIELD OF THE INVENTION

This invention pertains to the field of analyte determination by massanalysis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the reaction of an analyte with two differentisobaric labeling reagents (e.g. compounds I and II).

FIG. 1B illustrates the fragmentation of the labeled analyte illustratedin FIG. 1A to thereby produce reporter moieties (e.g. compounds VII andVIII as signature ions) of different masses from the isobaricallylabeled analytes.

FIG. 2 is an expansion plot of a mass spectrum of a labeled analyte.

FIG. 3 is the complete mass spectrum obtained from a second massanalysis of the selected labeled analyte identified in the expansionplot of FIG. 2.

FIG. 4 is an expansion plot of a mass spectrum of the predominate y-iondaughter fragment ion of the analyte as determined in the second massanalysis.

FIG. 5 is an expansion plot of a mass spectrum of the predominate b-iondaughter fragment ion of the analyte as determined in the second massanalysis.

FIG. 6 is an expansion plot of a mass spectrum of two reporters (i.e.signature ions) as determined in the second mass analysis.

FIG. 7 is a plot of observed vs. predicted ratios of reportersdetermined by a second mass analysis for various mixtures of a labeledpeptide, each peptide of the mixture comprising one of two differentreporters.

FIG. 8 is an illustration of two sets of isobaric labeling reagentswherein the same isotopes (compounds X-XM) and different isotopes(compounds XV-XVIII) are used within the set to thereby achievereporter/linker moieties of the same gross mass but each with a reportermoiety of a different gross mass within the set.

FIGS. 9A and 9B are an illustration of synthetic routes to isotopicallylabeled piperazine labeling reagents from basic starting materials. Theroute can also be used to prepare non-isotopically labeled piperazinereagents wherein non-isotopically labeled starting materials are used.

FIG. 10 is an illustration of a synthetic route to isotopically labeledand non-isotopically labeled N-alkyl piperazine labeling reagents frombasic starting materials.

FIG. 11 is an illustration of a synthetic route to isotopically labeledand non-isotopically labeled N-alkyl piperazine labeling reagents frombasic starting materials.

FIG. 12 is an illustration of a solid phase based synthetic route toisotopically labeled and non-isotopically labeled piperazine labelingreagents from basic starting materials.

1. INTRODUCTION

This invention pertains to methods, mixtures, kits and/or compositionsfor the determination of an analyte or analytes by mass analysis. Ananalyte can be any molecule of interest. Non-limiting examples ofanalytes include, but are not limited to, proteins, peptides, nucleicacids, carbohydrates, lipids, steroids and small molecules of less than1500 daltons.

Analytes can be labeled by reaction of the analyte with a labelingreagent of the formula: RP-X-LK-Y-RG, or a salt thereof, wherein RG is areactive group that reacts with the analyte and RP, X, LK and Y aredescribed in more detail below. A labeled analyte therefore can have thegeneral formula: RP-X-LK-Y-Analyte. Sets of isomeric or isobariclabeling reagents can be used to label the analytes of two or moredifferent samples wherein the labeling reagent can be different for eachdifferent sample and wherein the labeling reagent can comprise a uniquereporter, “RP”, that can be associated with the sample from which thelabeled analyte originated. Hence, information, such as the presenceand/or amount of the reporter, can be correlated with the presenceand/or amount (often expressed as a concentration and/or quantity) ofthe analyte in a sample even from the analysis of a complex mixture oflabeled analytes derived by mixing the products of the labeling ofdifferent samples. Analysis of such complex sample mixtures can beperformed in a manner that allows for the determination of one or aplurality of analytes from the same or from multiple samples in amultiplex manner. Thus, the methods, mixtures, kits and/or compositionsof this invention are particularly well suited for the multiplexanalysis of complex sample mixtures. For example, they can be used inproteomic analysis and/or genomic analysis as well as for correlationstudies related to genomic and proteomic analysis.

2. Definitions:

For the purposes of interpreting of this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa:

a. As used herein, “analyte” refers to a molecule of interest that maybe determined. Non-limiting examples of analytes can include, but arenot limited to, proteins, peptides, nucleic acids (both DNA or RNA),carbohydrates, lipids, steroids and/or other small molecules with amolecular weight of less than 1500 daltons. The source of the analyte,or the sample comprising the analyte, is not a limitation as it can comefrom any source. The analyte or analytes can be natural or synthetic.Non-limiting examples of sources for the analyte, or the samplecomprising the analyte, include but are not limited to cells or tissues,or cultures (or subcultures) thereof. Non-limiting examples of analytesources include, but are not limited to, crude or processed cell lysates(including whole cell lysates), body fluids, tissue extracts or cellextracts. Still other non-limiting examples of sources for the analyteinclude but are not limited to fractions from a separations process suchas a chromatographic separation or an electrophoretic separation. Bodyfluids include, but are not limited to, blood, urine, feces, spinalfluid, cerebral fluid, amniotic fluid, lymph fluid or a fluid from aglandular secretion. By processed cell lysate we mean that the celllysate is treated, in addition to the treatments needed to lyse thecell, to thereby perform additional processing of the collectedmaterial. For example, the sample can be a cell lysate comprising one ormore analytes that are peptides formed by treatment of the total proteincomponent of a crude cell lysate with a proteolytic enzyme to therebydigest precursor protein or proteins.

b. As used herein, “fragmentation” refers to the breaking of a covalentbond.

c. As used herein, “fragment” refers to a product of fragmentation(noun) or the operation of causing fragmentation (verb).

d. It is well accepted that the mass of an atom or molecule can beapproximated, often to the nearest whole number atomic mass unit or thenearest tenth or hundredth of an atomic mass unit. As used herein,“gross mass” refers to the absolute mass as well as to the approximatemass within a range where the use of isotopes of different atom typesare so close in mass that they are the functional equivalent for thepurpose of balancing the mass of the reporter and/or linker moieties (sothat the gross mass of the reporter/linker combination is the samewithin a set or kit of isobaric or isomeric labeling reagents) whetheror not the very small difference in mass of the different isotopes typesused can be detected.

For example, the common isotopes of oxygen have a gross mass of 16.0(actual mass 15.9949) and 18.0 (actual mass 17.9992), the commonisotopes of carbon have a gross mass of 12.0 (actual mass 12.00000) and13.0 (actual mass 13.00336) and the common isotopes of nitrogen have agross mass of 14.0 (actual mass 14.0031) and 15.0 (actual mass 15.0001).Whilst these values are approximate, one of skill in the art willappreciate that if one uses the ¹⁸O isotope in one reporter of a set,the additional 2 mass units (over the isotope of oxygen having a grossmass of 16.0) can, for example, be compensated for in a differentreporter of the set comprising ¹⁶O by incorporating, elsewhere in thereporter, two carbon ¹³C atoms, instead of two ¹²C atoms, two ¹⁵N atoms,instead of two ¹⁴N atoms or even one ¹³C atom and one ¹⁵N atom, insteadof a ¹²C and a ¹⁴N, to compensate for the ¹⁸O. In this way the twodifferent reporters of the set are the functional mass equivalent (i.e.have the same gross mass) since the very small actual differences inmass between the use of two ¹³C atoms (instead of two ¹²C atoms), two¹⁵N atoms (instead of two ¹⁴N atoms), one ¹³C and one ¹⁵N (instead of a¹²C and ¹⁴N) or one ¹⁸O atom (instead of one ¹⁶O atom), to therebyachieve an increase in mass of two Daltons, in all of the labels of theset or kit, is not an impediment to the nature of the analysis.

This can be illustrated with reference to FIG. 8. In FIG. 8, thereporter/linker combination of compound XVII (FIG. 8; chemical formula:C₅ ¹³CH₁₀ ¹⁵N₂O) has two ¹⁵N atoms and one ¹³C atom and a totaltheoretical mass of 129.138. By comparison, isobar XV (FIG. 8; chemicalformula C₅ ¹³CH₁₀N₂ ¹⁸O) has one ¹⁸O atom and one ¹³C atom and a totaltheoretical mass of 129.151. Compounds XVII and XV are isobars that arestructurally and chemically indistinguishable, except for heavy atomisotope content, although there is a slight absolute mass difference(mass 129.138 vs. mass 129.151 respectively). However, the gross mass ofcompounds XVII and XV is 129.1 for the purposes of this invention sincethis is not an impediment to the analysis whether or not the massspectrometer is sensitive enough to measure the small difference betweenthe absolute mass of isobars XVII and XV.

From FIG. 8, it is clear that the distribution of the same heavy atomisotopes within a structure is not the only consideration for thecreation of sets of isomeric and/or isobaric labeling reagents. It ispossible to mix heavy atom isotope types to achieve isomers or isobarsof a desired gross mass. In this way, both the selection (combination)of heavy atom isotopes as well as their distribution is available forconsideration in the production of the isomeric and/or isobaric labelingreagents useful for embodiments of this invention.

e. As used herein, “isotopically enriched” refers to a compound (e.g.labeling reagent) that has been enriched synthetically with one or moreheavy atom isotopes (e.g. stable isotopes such as Deuterium, ¹³C, ¹⁵N,¹⁸O, ³⁷Cl or ⁸¹Br). Because isotopic enrichment is not 100% effective,there can be impurities of the compound that are of lesser states ofenrichment and these will have a lower mass. Likewise, because ofover-enrichment (undesired enrichment) and because of natural isotopicabundance, there can be impurities of greater mass.

f. As used herein, “labeling reagent” refers to a moiety suitable tomark an analyte for determination. The term label is synonymous with theterms tag and mark and other equivalent terms and phrases. For example,a labeled analyte can also be referred to as a tagged analyte or amarked analyte. Accordingly the terms “label”, “tag”, “mark” andderivatives of these terms, are interchangeable and refer to a moietysuitable to mark, or that has marked, an analyte for determination.

g. As used herein, “support”, “solid support” or “solid carrier” meansany solid phase material upon which a labeling reagent can beimmobilized. Immobilization can, for example, be used to label analytesor be used to prepare a labeling reagent, whether or not the labelingoccurs on the support. Solid support encompasses terms such as “resin”,“synthesis support”, “solid phase”, “surface” “membrane” and/or“support”. A solid support can be composed of organic polymers such aspolystyrene, polyethylene, polypropylene, polyfluoroethylene,polyethyleneoxy, and polyacrylamide, as well as co-polymers and graftsthereof. A solid support can also be inorganic, such as glass, silica,controlled-pore-glass (CPG), or reverse-phase silica. The configurationof a solid support can be in the form of beads, spheres, particles,granules, a gel, a membrane or a surface. Surfaces can be planar,substantially planar, or non-planar. Solid supports can be porous ornon-porous, and can have swelling or non-swelling characteristics. Asolid support can be configured in the form of a well, depression orother container, vessel, feature or location. A plurality of solidsupports can be configured in an array at various locations, addressablefor robotic delivery of reagents, or by detection methods and/orinstruments.

h. As used herein, “natural isotopic abundance” refers to the level (ordistribution) of one or more isotopes found in a compound based upon thenatural prevalence of an isotope or isotopes in nature. For example, anatural compound obtained from living plant matter will typicallycontain about 0.6% ¹³C.

3. General:

The Reactive Group:

The reactive group “RG” of the labeling reagent or reagents used in themethod, mixture, kit and/or composition embodiments can be either anelectrophile or a nucleophile that is capable of reacting with one ormore reactive analytes of a sample. The reactive group can bepreexisting or it can be prepared in-situ. In-situ preparation of thereactive group can proceed in the absence of the reactive analyte or itcan proceed in the presence of the reactive analyte. For example, acarboxylic acid group can be modified in-situ with water-solublecarbodiimide (e.g. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride; EDC) to thereby prepare an electrophilic group that canbe reacted with a nucleophile such as an amine group. In someembodiments, activation of the carboxylic acid group of a labelingreagent with EDC can be performed in the presence of an amine(nucleophile) containing analyte. In some embodiments, the amine(nucleophile) containing analyte can also be added after the initialreaction with EDC is performed. In other embodiments, the reactive groupcan be generated in-situ by the in-situ removal of a protecting group.Consequently, any existing or newly created reagent or reagents that caneffect the derivatization of analytes by the reaction of nucleophilesand/or electrophiles are contemplated by the method, mixture, kit and/orcomposition embodiments of this invention.

Where the reactive group of the labeling reagent is an electrophile, itcan react with a suitable nucleophilic group of the analyte or analytes.Where the reactive group of the labeling reagent is a nucleophile, itcan react with a suitable electrophilic group of the analyte oranalytes. Numerous pairs of suitable nucleophilic groups andelectrophilic groups are known and often used in the chemical andbiochemical arts. Non-limiting examples of reagents comprising suitablenucleophilic or electrophilic groups that can be coupled to analytes(e.g. such as proteins, peptides, nucleic acids, carbohydrates, lipids,steroids or other small molecules of less that 1500 daltons) to effecttheir derivatization, are described in the Pierce Life Science &Analytical Research Products Catalog & Handbook (a Perstorp BiotecCompany), Rockford, Ill. 61105, USA. Other suitable reagents are wellknown in the art and are commercially available from numerous othervendors such as Sigma-Aldrich.

The reactive group of a labeling reagent can be an amine reactive group.For example the amine reactive group can be an active ester. Activeesters are well known in peptide synthesis and refer to certain estersthat are easily reacted with the N-α amine of an amino acid underconditions commonly used in peptide synthesis. The amine reactive activeester can be an N-hydroxysuccinimidyl ester, aN-hydroxysulfosuccinimidyl ester, a pentafluorophenyl ester, a2-nitrophenyl ester, a 4-nitrophenyl ester, a 2,4-dinitrophenylester ora 2,4-dihalophenyl ester. For example, the alcohol or thiol group of anactive ester can have the formula:

wherein X is O or S, but preferably O. All of the foregoing beingalcohol or thiol groups known to form active esters in the field ofpeptide chemistry wherein said alcohol or thiol group is displaced bythe reaction of the N-α-amine of the amino acid with the carbonyl carbonof the ester. It should be apparent that the active ester (e.g.N-hydroxysuccinimidyl ester) of any suitable labelling/tagging reagentdescribed herein could be prepared using well-known procedures (See:Greg T. Hermanson (1996). “The Chemistry of Reactive Groups” in“Bioconjugate Techniques” Chapter 2 pages 137-165, Academic Press, (NewYork); also see: Innovation And Perspectives In Solid Phase Synthesis,Editor: Roger Epton, SPCC (UK) Ltd, Birmingham, 1990). Methods for theformation of active esters of N-substituted piperazine acetic acidscompounds that are representative examples of labelling reagents of thegeneral formula: RP-X-LK-Y-RG, are described in co-pending and commonlyowned U.S. patent application Ser. No. 10/751,354, incorporated hereinby reference.

In another embodiment, the reactive group of the labeling reagent can bea mixed anhydride since mixed anhydrides are known to efficiently reactwith amine groups to thereby produce amide bonds.

The reactive group of a labeling reagent can be a thiol reactive group.For example, the thiol reactive group can be a malemide, an alkylhalide, an aryl halide of an α-halo-acyl. By halide or halo we meanatoms of fluorine, chlorine, bromine or iodine.

The reactive group of a labeling reagent can be a hydroxyl reactivegroup. For example, the hydroxyl reactive group can be a trityl-halideor a silyl-halide reactive moiety. The trityl-halide reactive moietiescan be substituted (e.g. Y-methoxytrityl, Y-dimethoxytrityl,Y-trimethoxytrityl, etc) or unsubstituted wherein Y is defined below.The silyl reactive moieties can be alkyl substituted silyl halides, suchas Y-dimethylsilyl, Y-ditriethylsilyl, Y-dipropylsilyl,Y-diisopropylsilyl, etc.) wherein Y is defined below.

The reactive group of the labeling reagent can be a nucleophile such asan amine group, a hydroxyl group or a thiol group.

The Reporter Moiety:

The reporter moiety of the labeling reagent or reagents used in themethod, mixture, kit and/or composition embodiments is a group that hasa unique mass (or mass to charge ratio) that can be determined.Accordingly, each reporter of a set can have a unique gross mass.Different reporters can comprise one or more heavy atom isotopes toachieve their unique mass. For example, isotopes of carbon (¹²C, ¹³C and¹⁴C), nitrogen (¹⁴N and ¹⁵N), oxygen (¹⁶O and ¹⁸O) or hydrogen(hydrogen, deuterium and tritium) exist and can be used in thepreparation of a diverse group of reporter moieties. Examples of stableheavy atom isotopes include ¹³C, ¹⁵N, ¹⁸O and deuterium. These are notlimiting as other light and heavy atom isotopes can also be used in thereporter. Basic starting materials suitable for preparing reporterscomprising light and heavy atom isotopes are available from variouscommercial sources such as Cambridge Isotope Laboratories, Andover,Mass. (See: list or “basic starting materials” at www.isotope.com) andIsotec (a division of Sigma-Aldrich). Cambridge Isotope Laboratories andIsotec will also prepare desired compounds under custom synthesiscontracts. Id.

A unique reporter can be associated with a sample of interest therebylabeling one or multiple analytes of that sample with the reporter. Inthis way information about the reporter can be associated withinformation about one or all of the analytes of the sample. However, thereporter need not be physically linked to an analyte when the reporteris determined. Rather, the unique gross mass of the reporter can, forexample, be determined in a second mass analysis of a tandem massanalyzer, after ions of the labeled analyte are fragmented to therebyproduce daughter fragment ions and detectable reporters. The determinedreporter can be used to identify the sample from which a determinedanalyte originated. Further, the amount of the unique reporter, eitherrelative to the amount of other reporters or relative to a calibrationstandard (e.g. an analyte labeled with a specific reporter), can be usedto determine the relative or absolute amount (often expressed as aconcentration and/or quantity) of analyte in the sample or samples.Therefore information, such as the amount of one or more analytes in aparticular sample, can be associated with the reporter moiety that isused to label each particular sample. Where the identity of the analyteor analytes is also determined, that information can be correlated withinformation pertaining to the different reporters to thereby facilitatethe determination of the identity and amount of each labeled analyte inone or a plurality of samples.

The reporter either comprises a fixed charge or is capable of becomingionized. Because the reporter either comprises a fixed charge or iscapable of being ionized, the labeling reagent might be isolated or usedto label the reactive analyte in a salt or zwitterionic form. Ionizationof the reporter facilitates its determination in a mass spectrometer.Accordingly, the reporter can be determined as an ion, sometimesreferred to as a signature ion. When ionized, the reporter can compriseone or more net positive or negative charges. Thus, the reporter cancomprise one or more acidic groups or basic groups since such groups canbe easily ionized in a mass spectrometer. For example, the reporter cancomprise one or more basic nitrogen atoms (positive charge) or one ormore ionizable acidic groups such as a carboxylic acid group, sulfonicacid group or phosphoric acid group (negative charge). Non-limitingexamples of reporters comprising a basic nitrogen include, substitutedor unsubstituted, morpholines, piperidines or piperazines.

The reporter can be a 5, 6 or 7 membered heterocyclic ring comprising aring nitrogen atom that is N-alkylated with a substituted orunsubstituted acetic acid moiety to which the analyte is linked throughthe carbonyl carbon of the N-alkyl acetic acid moiety, wherein eachdifferent label comprises one or more heavy atom isotopes. Theheterocyclic ring can be substituted or unsubstituted. The heterocyclicring can be aliphatic or aromatic. Possible substituents of theheterocylic moiety include alkyl, alkoxy and aryl groups. Thesubstituents can comprise protected or unprotected groups, such asamine, hydroxyl or thiol groups, suitable for linking the analyte to asupport. The heterocyclic ring can comprise additional heteroatoms suchas one or more nitrogen, oxygen or sulfur atoms.

The reporter can be selected so that it does not substantiallysub-fragment under conditions typical for the analysis of the analyte.The reporter can be chosen so that it does not substantiallysub-fragment under conditions of dissociative energy applied to causefragmentation of both bonds X and Y of at least a portion of selectedions of a labeled analyte in a mass spectrometer. By “does notsubstantially sub-fragment” we mean that fragments of the reporter aredifficult or impossible to detect above background noise when applied tothe successful analysis of the analyte of interest. The gross mass of areporter can be intentionally selected to be different as compared withthe mass of the analyte sought to be determined or any of the expectedfragments of the analyte. For example, where proteins or peptides arethe analytes, the reporter's gross mass can be chosen to be different ascompared with any naturally occurring amino acid or peptide, or expectedfragments thereof. This can facilitate analyte determination since,depending on the analyte, the lack of any possible components of thesample having the same coincident mass can add confidence to the resultof any analysis.

The reporter can be a small molecule that is non-polymeric. The reporterdoes not have to be a biopolymer (e.g. a peptide, a protein or a nucleicacid) or a component of a biopolymer (e.g. an amino acid, a nucleosideor a nucleotide). The gross mass of a reporter can be less than 250Daltons. Such a small molecule can be easily determined in the secondmass analysis, free from other components of the sample having the samecoincident mass in the first mass analysis. In this context, the secondmass analysis can be performed, typically in a tandem mass spectrometer,on selected ions that are determined in the first mass analysis. Becauseions of a particular mass to charge ratio can be specifically selectedout of the first mass analysis for possible fragmentation and furthermass analysis, the non-selected ions from the first mass analysis arenot carried forward to the second mass analysis and therefore do notcontaminate the spectrum of the second mass analysis. Furthermore, thesensitivity of a mass spectrometer and the linearity of the detector(for purposes of quantitation) can be quite robust in this low massrange. Additionally, the present state of mass spectrometer technologycan allow for baseline mass resolution of less than one Dalton in thismass range (See for example: FIG. 6). These factors may prove to beuseful advancements to the state of the art.

The Linker Moiety:

The linker moiety of the labeling reagent or reagents used with themethod, mixture, kit and/or composition embodiments links the reporterto the analyte or the reporter to the reactive group depending onwhether or not a reaction with the analyte has occurred. The linker canbe selected to produce a neutral species when both bonds X and Y arefragmented (i.e. undergoes neutral loss upon fragmentation of both bondsX and Y). The linker can be a very small moiety such as a carbonyl orthiocarbonyl group. For example, the linker can comprise at least oneheavy atom isotope and comprise the formula:

wherein R¹ is the same or different and is an alkyl group comprising oneto eight carbon atoms which may optionally contain a heteroatom or asubstituted or unsubstituted aryl group wherein the carbon atoms of thealkyl and aryl groups independently comprise linked hydrogen, deuteriumand/or fluorine atoms. The linker can be a larger moiety. The linker canbe a polymer or a biopolymer. The linker can be designed to sub-fragmentwhen subjected to dissociative energy levels; includingsub-fragmentation to thereby produce only neutral fragments of thelinker.

The linker moiety can comprise one or more heavy atom isotopes such thatits mass compensates for the difference in gross mass between thereporters for each labeled analyte of a mixture or for the reagents ofset and/or kit. Moreover, the aggregate gross mass (i.e. the gross masstaken as a whole) of the reporter/linker combination can be the same foreach labeled analyte of a mixture or for the reagents of set and/or kit.More specifically, the linker moiety can compensate for the differencein gross mass between reporters of labeled analytes from differentsamples wherein the unique gross mass of the reporter correlates withthe sample from which the labeled analyte originated and the aggregategross mass of the reporter/linker combination is the same for eachlabeled analyte of a sample mixture regardless of the sample from whichit originated. In this way, the gross mass of identical analytes in twoor more different samples can have the same gross mass when labeled andthen mixed to produce a sample mixture.

For example, the labeled analytes, or the reagents of a set and/or kitfor labeling the analytes, can be isomers or isobars. Thus, if ions of aparticular mass to charge ratio (taken from the sample mixture) areselected (i.e. selected ions) in a mass spectrometer from an initialmass analysis of the sample mixture, identical analytes from thedifferent samples that make up the sample mixture are represented in theselected ions in proportion to their respective concentration and/orquantity in the sample mixture. Accordingly, the linker not only linksthe reporter to the analyte, it also can serve to compensate for thediffering masses of the unique reporter moieties to thereby harmonizethe gross mass of the reporter/linker combination in the labeledanalytes of the various samples.

Because the linker can act as a mass balance for the reporter in thelabeling reagents, such that the aggregate gross mass of thereporter/linker combination is the same for all reagents of a set orkit, the greater the number of atoms in the linker, the greater thepossible number of different isomeric/isobaric labeling reagents of aset and/or kit. Stated differently, generally the greater the number ofatoms that a linker comprises, the greater number of potentialreporter/linker combinations exist since isotopes can be substituted atmost any position in the linker to thereby produce isomers or isobars ofthe linker portion wherein the linker portion is used to offset thediffering masses of the reporter portion and thereby create a set ofreporter/linker isomers or isobars. Such diverse sets of labelingreagents are particularly well suited for multiplex analysis of analytesin the same and/or different samples.

The total number of labeling reagents of a set and/or kit can be two,three, four, five, six, seven, eight, nine, ten or more. The diversityof the labeling reagents of a set or kit is limited only by the numberof atoms of the reporter and linker moieties, the heavy atom isotopesavailable to substitute for the light isotopes and the various syntheticconfigurations in which the isotopes can be synthetically placed. Assuggested above however, numerous isotopically enriched basic startingmaterials are readily available from manufacturers such as CambridgeIsotope Laboratories and Isotec. Such isotopically enriched basicstarting materials can be used in the synthetic processes used toproduce sets of isobaric and isomeric labeling reagents or be used toproduce the isotopically enriched starting materials that can be used inthe synthetic processes used to produce sets of isobaric and isomericlabeling reagents. Some examples of the preparation of isobaric labelingreagents suitable for use in a set of labeling reagents can be found inthe Examples section, below.

The Reporter/Linker Combination:

The labeling reagents described herein comprise reporters and linkersthat are linked through the bond X. As described above, thereporter/linker combination can be identical in gross mass for eachmember of a set and/or kit of labeling reagents. Moreover, bond X of thereporter/linker combination of the labeling reagents can be designed tofragment, in at least a portion of the selected ions, when subjected todissociative energy levels thereby releasing the reporter from theanalyte. Accordingly, the gross mass of the reporter (as a m/s ratio)and its intensity can be observed directly in MS/MS analysis.

The reporter/linker combination can comprise various combinations of thesame or different heavy atom isotopes amongst the various labelingreagents of a set or kit. In the scientific literature this hassometimes been referred to as coding or isotope coding. For example,Abersold et al. has disclosed the isotope coded affinity tag (ICAT; seeWO00/11208). In one respect, the reagents of Abersold et al. differ fromthe labeling reagents of this invention in that Abersold does not teachtwo or more same mass labeling reagents such as isomeric or isobariclabeling reagents.

Mass Spectrometers/Mass Spectrometry (MS):

The methods of this invention can be practiced using tandem massspectrometers and other mass spectrometers that have the ability toselect and fragment molecular ions. Tandem mass spectrometers (and to alesser degree single-stage mass spectrometers) have the ability toselect and fragment molecular ions according to their mass-to-charge(m/z) ratio, and then record the resulting fragment (daughter) ionspectra. More specifically, daughter fragment ion spectra can begenerated by subjecting selected ions to dissociative energy levels(e.g. collision-induced dissociation (CID)). For example, ionscorresponding to labeled peptides of a particular m/z ratio can beselected from a first mass analysis, fragmented and reanalyzed in asecond mass analysis. Representative instruments that can perform suchtandem mass analysis include, but are not limited to, magneticfour-sector, tandem time-of-flight, triple quadrupole, ion-trap, andhybrid quadrupole time-of-flight (Q-TOF) mass spectrometers.

These types of mass spectrometers may be used in conjunction with avariety of ionization sources, including, but not limited to,electrospray ionization (ESI) and matrix-assisted laser desorptionionization (MALDI). Ionization sources can be used to generate chargedspecies for the first mass analysis where the analytes do not alreadypossess a fixed charge. Additional mass spectrometry instruments andfragmentation methods include post-source decay in MALDI-MS instrumentsand high-energy CID using MALDI-TOF(time of ffight)-TOF MS. For a recentreview of tandem mass spectrometers please see: R. Aebersold and D.Goodlett, Mass Spectrometry in Proteomics. Chem. Rev. 101: 269-295(2001). Also see U.S. Pat. No. 6,319,476, herein incorporated byreference, for a discussion of TOF-TOF mass analysis techniques.

Fragmentation by Dissociative Energy Levels:

It is well accepted that bonds can fragment as a result of the processesoccurring in a mass spectrometer. Moreover, bond fragmentation can beinduced in a mass spectrometer by subjecting ions to dissociative energylevels. For example, the dissociative energy levels can be produced in amass spectrometer by collision-induced dissociation (CID). Those ofordinary skill in the art of mass spectrometry will appreciate thatother exemplary techniques for imposing dissociative energy levels thatcause fragmentation include, but are not limited to, photo dissociation,electron capture and surface induced dissociation.

The process of fragmenting bonds by collision-induced dissociationinvolves increasing the kinetic energy state of selected ions, throughcollision with an inert gas, to a point where bond fragmentation occurs.For example, kinetic energy can be transferred by collision with aninert gas (such as nitrogen, helium or argon) in a collision cell. Theamount of kinetic energy that can be transferred to the ions isproportional to the number of gas molecules that are allowed to enterthe collision cell. When more gas molecules are present, a greateramount of kinetic energy can be transferred to the selected ions, andless kinetic energy is transferred when there are fewer gas moleculespresent.

It is therefore clear that the dissociative energy level in a massspectrometer can be controlled. It is also well accepted that certainbonds are more labile than other bonds. The lability of the bonds in ananalyte or the reporter/linker moiety depends upon the nature of theanalyte or the reporter/linker moiety. Accordingly, the dissociativeenergy levels can be adjusted so that the analytes and/or the labels(e.g. the reporter/linker combinations) can be fragmented in a mannerthat is determinable. One of skill in the art will appreciate how tomake such routine adjustments to the components of a mass spectrometerto thereby achieve the appropriate level of dissociative energy tothereby fragment at least a portion of ions of labeled analytes intoionized reporter moieties and daughter fragment ions.

For example, dissociative energy can be applied to ions that areselected/isolated from the first mass analysis. In a tandem massspectrometer, the extracted ions can be subjected to dissociative energylevels and then transferred to a second mass analyzer. The selected ionscan have a selected mass to charge ratio. The mass to charge ratio canbe within a range of mass to charge ratios depending upon thecharacteristics of the mass spectrometer. When collision induceddissociation is used, the ions can be transferred from the first to thesecond mass analyzer by passing them through a collision cell where thedissociative energy can be applied to thereby produce fragment ions. Forexample the ions sent to the second mass analyzer for analysis caninclude some, or a portion, of the remaining (unfragmented) selectedions, as well as reporter ions (signature ions) and daughter fragmentions of the labeled analyte.

Analyte Determination by Computer Assisted Database Analysis:

In some embodiments, analytes can be determined based upon daughter-ionfragmentation patterns that are analyzed by computer-assisted comparisonwith the spectra of known or “theoretical” analytes. For example, thedaughter fragment ion spectrum of a peptide ion fragmented underconditions of low energy CID can be considered the sum of many discretefragmentation events. The common nomenclature differentiates daughterfragment ions according to the amide bond that breaks and the peptidefragment that retains charge following bond fission. Charge-retention onthe N-terminal side of the fissile amide bond results in the formationof a b-type ion. If the charge remains on the C-terminal side of thebroken amide bond, then the fragment ion is referred to as a y-type ion.In addition to b- and y-type ions, the CID mass spectrum may containother diagnostic fragment ions (daughter fragment ions). These includeions generated by neutral loss of ammonia (−17 amu) from glutamine,lysine and arginine or the loss of water (−18 amu) fromhydroxyl-containing amino acids such as serine and threonine. Certainamino acids have been observed to fragment more readily under conditionsof low-energy CID than others. This is particularly apparent forpeptides containing proline or aspartic acid residues, and even more soat aspartyl-proline bonds (Mak, M. et al., Rapid Commun. Mass Spectrom.,12: 837-842) (1998). Accordingly, the peptide bond of a Z-pro dimer orZ-asp dimer, wherein Z is any natural amino acid, pro is proline and aspis aspartic acid, will tend to be more labile as compared with thepeptide bond between all other amino acid dimer combinations.

For peptide and protein samples therefore, low-energy CID spectracontain redundant sequence-specific information in overlapping b- andy-series ions, internal fragment ions from the same peptide, andimmonium and other neutral-loss ions. Interpreting such CID spectra toassemble the amino acid sequence of the parent peptide de novo ischallenging and time-consuming. The most significant advances inidentifying peptide sequences have been the development of computeralgorithms that correlate peptide CID spectra with peptide sequencesthat already exist in protein and DNA sequence databases. Suchapproaches are exemplified by programs such as SEQUEST (Eng, J. et al.J. Am. Soc. Mass Spectrom., 5: 976-989 (1994)) and MASCOT (Perkins, D.et al. Electrophoresis, 20: 3551-3567 (1999)).

In brief, experimental peptide CID spectra (MS/MS spectra) are matchedor correlated with ‘theoretical’ daughter fragment ion spectracomputationally generated from peptide sequences obtained from proteinor genome sequence databases. The match or correlation is based upon thesimilarities between the expected mass and the observed mass of thedaughter fragment ions in MS/MS mode. The potential match or correlationis scored according to how well the experimental and ‘theoretical’fragment patterns coincide. The constraints on databases searching for agiven peptide amino acid sequence are so discriminating that a singlepeptide CID spectrum can be adequate for identifying any given proteinin a whole-genome or expressed sequence tag (EST) database. For otherreviews please see: Yates, J. R. Trends, Genetics, 16: 5-8 (2000) andYates, J. R., Electrophoresis 19: 893-900 (1998).

Accordingly, daughter fragment ion analysis of MS/MS spectra can be usednot only to determine the analyte of a labeled analyte, it can also beused to determine analytes from which the determined analyte originated.For example, identification of a peptide in the MS/MS analysis can becan be used to determine the protein from which the peptide was cleavedas a consequence of an enzymatic digestion of the protein. It isenvisioned that such analysis can be applied to other analytes, such asnucleic acids.

Bonds X and Y:

X is a bond between an atom of the reporter and an atom of the linker. Yis a bond between an atom of the linker and an atom of either thereactive group or, if the labeling reagent has been reacted with areactive analyte, the analyte. Bonds X and Y of the various labelingreagents (i.e. RP-X-LK-Y-RG) that can be used in the embodiments of thisinvention can fragment, in at least a portion of selected ions, whensubjected to dissociative energy levels. Therefore, the dissociativeenergy level can be adjusted in a mass spectrometer so that both bonds Xand Y fragment in at least a portion of the selected ions of the labeledanalytes (i.e. RP-X-LK-Y-Analyte). Fragmentation of bond X releases thereporter from the analyte so that the reporter can be determinedindependently from the analyte. Fragmentation of bond Y releases thereporter/linker combination from the analyte, or the linker from theanalyte, depending on whether or not bond X has already been fragmented.Bond Y can be more labile than bond X. Bond X can be more labile thanbond Y. Bonds X and Y can be of the same relative lability.

When the analyte of interest is a protein or peptide, the relativelability of bonds X and Y can be adjusted with regard to an amide(peptide) bond. Bond X, bond Y or both bonds X and Y can be more, equalor less labile as compared with a typical amide (peptide) bond. Forexample, under conditions of dissociative energy, bond X and/or bond Ycan be less prone to fragmentation as compared with the peptide bond ofa Z-pro dimer or Z-asp dimer, wherein Z is any natural amino acid, prois proline and asp is aspartic acid. In some embodiments, bonds X and Ywill fragment with approximately the same level of dissociative energyas a typical amide bond. In some embodiments, bonds X and Y willfragment at a greater level of dissociative energy as compared with atypical amide bond.

Bonds X and Y can also exist such that fragmentation of bond Y resultsin the fragmentation of bond X, and vice versa. In this way, both bondsX and Y can fragment essentially simultaneously such that no substantialamount of analyte, or daughter fragment ion thereof, comprises a partiallabel in the second mass analysis. By “substantial amount of analyte” wemean that less than 25%, and preferably less than 10%, partially labeledanalyte can be determined in the MS/MS spectrum.

Because there can be a clear demarcation between labeled and unlabeledfragments of the analyte in the spectra of the second mass analysis(MS/MS), this feature can simplify the identification of the analytesfrom computer assisted analysis of the daughter fragment ion spectra.Moreover, because the fragment ions of analytes can, in someembodiments, be either fully labeled or unlabeled (but not partiallylabeled) with the reporter/linker moiety, there can be little or noscatter in the masses of the daughter fragment ions caused by isotopicdistribution across fractured bonds such as would be the case whereisotopes were present on each side of a single labile bond of apartially labeled analyte routinely determined in the second massanalysis.

Sample Processing:

In certain embodiments of this invention, a sample can be processedprior to, as well as after, labeling of the analytes. The processing canfacilitate the labeling of the analytes. The processing can facilitatethe analysis of the sample components. The processing can simplify thehandling of the samples. The processing can facilitate two or more ofthe foregoing.

For example, a sample can be treated with an enzyme. The enzyme can be aprotease (to degrade proteins and peptides), a nuclease (to degradenucleic acids) or some other enzyme. The enzyme can be chosen to have avery predictable degradation pattern. Two or more proteases and/or twoor more nuclease enzymes may also be used together, or with otherenzymes, to thereby degrade sample components.

For example, the proteolytic enzyme trypsin is a serine protease thatcleaves peptide bonds between lysine or arginine and an unspecific aminoacid to thereby produce peptides that comprise an amine terminus(N-terminus) and lysine or arginine carboxyl terminal amino acid(C-terminus). In this way the peptides from the cleavage of the proteinare predictable and their presence and/or quantity, in a sample from atrpsin digest, can be indicative of the presence and/or quantity of theprotein of their origin. Moreover, the free amine termini of a peptidecan be a good nucleophile that facilitates its labeling. Other exemplaryproteolytic enzymes include papain, pepsin, ArgC, LysC, V8 protease,AspN, pronase, chymotrypsin and carboxypeptidase C.

For example, a protein (e.g. protein Z) might produce three peptides(e.g. peptides B, C and D) when digested with a protease such astrypsin. Accordingly, a sample that has been digested with a proteolyticenzyme, such as trypsin, and that when analyzed is confirmed to containpeptides B, C and D, can be said to have originally comprised theprotein Z. The quantity of peptides B, C and D will also correlate withthe quantity of protein Z in the sample that was digested. In this way,any determination of the identity and/or quantify of one or more ofpeptides B, C and D in a sample (or a fraction thereof), can be used toidentify and/or quantify protein Z in the original sample (or a fractionthereof).

Because activity of the enzymes is predictable, the sequence of peptidesthat are produced from degradation of a protein of known sequence can bepredicted. With this information, “theoretical” peptide information canbe generated. A determination of the “theoretical” peptide fragments incomputer assisted analysis of daughter fragment ions (as describedabove) from mass spectrometry analysis of an actual sample can thereforebe used to determine one or more peptides or proteins in one or moreunknown samples.

Separation of the Sample Mixture:

In some embodiments the processing of a sample or sample mixture oflabeled analytes can involve separation. For example, a sample mixturecomprising differentially labeled analytes from different samples can beprepared. By differentially labeled we mean that each of the labelscomprises a unique property that can be identified (e.g. comprises aunique reporter moiety that produces a unique “signature ion” in MS/MSanalysis). In order to analyze the sample mixture, components of thesample mixture can be separated and mass analysis performed on only afraction of the sample mixture. In this way, the complexity of theanalysis can be substantially reduced since separated analytes can beindividually analyzed for mass thereby increasing the sensitivity of theanalysis process. Of course the analysis can be repeated one or moretime on one or more additional fractions of the sample mixture tothereby allow for the analysis of all fractions of the sample mixture.

Separation conditions under which identical analytes that aredifferentially labeled co-elute at a concentration, or in a quantity,that is in proportion to their abundance in the sample mixture can beused to determine the amount of each labeled analyte in each of thesamples that comprise the sample mixture provided that the amount ofeach sample added to the sample mixture is known. Accordingly, in someembodiments, separation of the sample mixture can simplify the analysiswhilst maintaining the correlation between signals determined in themass analysis (e.g. MS/MS analysis) with the amount of the differentlylabeled analytes in the sample mixture.

The separation can be performed by chromatography. For example, liquidchromatography/mass spectrometry (LC/MS) can be used to effect such asample separation and mass analysis. Moreover, any chromatographicseparation process suitable to separate the analytes of interest can beused. For example, the chromatographic separation can be normal phasechromatography, reversed-phase chromatography, ion-exchangechromatography, size exclusion chromatography or affinitychromatorgraphy.

The separation can be performed electrophoretically. Non-limitingexamples of electrophoretic separations techniques that can be usedinclude, but are not limited to, 1D electrophoretic separation, 2Delectrophoretic separation and/or capillary electrophoretic separation.

An isobaric labeling reagent or a set of reagents can be used to labelthe analytes of a sample. Isobaric labeling reagents are particularlyuseful when a separation step is performed because the isobaric labelsof a set of labeling reagents are structurally and chemicallyindistinguishable (and can be indistinguishable by gross mass untilfragmentation removes the reporter from the analyte). Thus, all analytesof identical composition that are labeled with different isobaric labelscan chromatograph in exactly the same manner (i.e. co-elute). Becausethey are structurally and chemically indistinguishable, the eluent fromthe separation process can comprise an amount of each isobaricallylabeled analyte that is in proportion to the amount of that labeledanalyte in the sample mixture. Furthermore, from the knowledge of howthe sample mixture was prepared (portions of samples, an other optionalcomponents (e.g. calibration standards) added to prepare the samplemixture), it is possible to relate the amount of labeled analyte in thesample mixture back to the amount of that labeled analyte in the samplefrom which it originated.

The labeling reagents can also be isomeric. Although isomers cansometimes be chromatographically separated, there are circumstances,that are condition dependent, where the separation process can beoperated to co-elute all of the identical analytes that aredifferentially labeled wherein the amount of all of the labeled analytesexist in the eluent in proportion to their concentration and/or quantityin the sample mixture.

As used herein, isobars differ from isomers in that isobars arestructurally and chemically indistinguishable compounds (except forisotopic content and/or distribution) of the same nominal gross mass(See for example, FIG. 1) whereas isomers are structurally and/orchemically distinguishable compounds of the same nominal gross mass.

Relative and Absolute Quantitation of Analytes:

In some embodiments, the relative quantitation of differentially labeledidentical analytes of a sample mixture is possible. Relativequantitation of differentially labeled identical analytes is possible bycomparison of the relative amounts of reporter (e.g. area or height ofthe peak reported) that are determined in the second mass analysis for aselected, labeled analyte observed in a first mass analysis. Putdifferently, where each reporter can be correlated with information fora particular sample used to produce a sample mixture, the relativeamount of that reporter, with respect to other reporters observed in thesecond mass analysis, is the relative amount of that analyte in thesample mixture. Where components combined to form the sample mixture areknown, the relative amount of the analyte in each sample used to preparethe sample mixture can be back calculated based upon the relativeamounts of reporter observed for the ions of the labeled analyteselected from the first mass analysis. This process can be repeated forall of the different labeled analytes observed in the first massanalysis. In this way, the relative amount (often expressed in terms ofconcentration and/or quantity) of each reactive analyte, in each of thedifferent samples used to produce the sample mixture, can be determined.

In other embodiments, absolute quantitation of analytes can bedetermined. For these embodiments, a known amount of one or moredifferentially labeled analytes (the calibration standard or calibrationstandards) can be added to the sample mixture. The calibration standardcan be an expected analyte that is labeled with an isomeric or isobariclabel of the set of labels used to label the analytes of the samplemixture provided that the reporter for the calibration standard isunique as compared with any of the samples used to form the samplemixture. Once the relative amount of reporter for the calibrationstandard, or standards, is determined with relation to the relativeamounts of the reporter for the differentially labeled analytes of thesample mixture, it is possible to calculate the absolute amount (oftenexpressed in concentration and/or quantity) of all of the differentiallylabeled analytes in the sample mixture. In this way, the absolute amountof each differentially labeled analyte (for which there is a calibrationstandard in the sample from which the analyte originated) can also bedetermined based upon the knowledge of how the sample mixture wasprepared.

Notwithstanding the foregoing, corrections to the intensity of thereporters (signature ions) can be made, as appropriate, for anynaturally occurring, or artificially created, isotopic abundance withinthe reporters. An example of such a correction can be found in Example3. A more sophisticated example of these types of corrections can alsobe found in copending and co-owned U.S. Provisional Patent ApplicationSer. No. 60/524,844, entitled: “Method and Apparatus For De-ConvolutingA Convoluted Spectrum”, filed on Nov. 26, 2003. The more care taken toaccurately quantify the intensity of each reporter, the more accuratewill be the relative and absolute quantification of the analytes in theoriginal samples.

Proteomic Analysis:

The methods, mixtures, kits and/or compositions of this invention can beused for complex analysis because samples can be multiplexed, analyzedand reanalyzed in a rapid and repetitive manner using mass analysistechniques. For example, sample mixtures can be analyzed for the amountof individual analytes in one or more samples. The amount (oftenexpressed in concentration and/or quantity) of those analytes can bedetermined for the samples from which the sample mixture was comprised.Because the sample processing and mass analyses can be performedrapidly, these methods can be repeated numerous times so that the amountof many differentially labeled analytes of the sample mixture can bedetermined with regard to their relative and/or absolute amounts in thesample from which the analyte originated.

One application where such a rapid multiplex analysis is useful is inthe area of proteomic analysis. Proteomics can be viewed as anexperimental approach to describe the information encoded in genomicsequences in terms of structure, function and regulation of biologicalprocesses. This may be achieved by systematic analysis of the totalprotein component expressed by a cell or tissue. Mass spectrometry, usedin combination with the method, mixture, kit and/or compositionembodiments of this invention is one possible tool for such globalprotein analysis.

For example, with a set of four isobaric labeling reagents, it ispossible to obtain four time points in an experiment to determine up ordown regulation of protein expression, for example, based upon responseof growing cells to a particular stimulant. It is also possible toperform fewer time points but to incorporate one or two controls. In allcases, up or down regulation of the protein expression, optionally withrespect to the controls, can be determined in a single multiplexexperiment. Moreover, because processing is performed in parallel theresults are directly comparable, since there is no risk that slightvariations in protocol may have affected the results.

4. Description of Various Embodiments of the Invention:

A. Methods

According to the methods of this invention, the analyte to be determinedis labeled. The labeled analyte, the analyte itself, one or morefragments of the analyte and/or fragments of the label, can bedetermined by mass analysis. In some embodiments, methods of thisinvention can be used for the analysis of different analytes in the samesample as well as for the multiplex analysis of the same and/ordifferent analytes in two or more different samples. The two or moresamples can be mixed to form a sample mixture. In the multiplexanalysis, labeling reagents can be used to determine from which sampleof a sample mixture an analyte originated. The absolute and/or relative(with respect to the same analyte in different samples) amount (oftenexpressed in concentration or quantity) of the analyte, in each of twoor more of the samples combined to form the sample mixture, can bedetermined. Moreover, the mass analysis of fragments of the analyte(e.g. daughter fragment ions) can be used to identify the analyte and/orthe precursor to the analyte; such as where the precursor molecule tothe analyte was degraded.

One distinction of the described approach lies in the fact that analytesfrom different samples can be differentially isotopically labeled (i.e.isotopically coded) with unique labels that are chemically isomeric orisobaric (have equal mass) and that identify the sample from which theanalyte originated. The differentially labeled analytes are notdistinguished in MS mode of a mass spectrometer because they all haveidentical (gross) mass to charge ratios. However, when subjected todissociative energy levels, such as through collision induceddissociation (CID), the labels can fragment to yield unique reportersthat can be resolved by mass (mass to charge ratio) in a massspectrometer. The relative amount of reporter observed in the massspectrum can correlate with the relative amount of a labeled analyte inthe sample mixture and, by implication, the amount of that analyte in asample from which it originated. Thus, the relative intensities of thereporters (i.e. signature ions) can be used to measure the relativeamount of an analyte or analytes in two or more different samples thatwere combined to form a sample mixture. From the reporter information,absolute amounts (often expressed as concentration and/or quantity) ofan analyte or analytes in two or more samples can be derived ifcalibration standards for the each analyte, for which absolutequantification is desired, are incorporated into the sample mixture.

For example, the analyte might be a peptide that resulted from thedegradation of a protein using an enzymatic digestion reaction toprocess the sample. Protein degradation can be accomplished by treatmentof the sample with a proteolytic enzyme (e.g. trypsin, papain, pepsin,ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin or carboxypeptidaseC). By determination of the identity and amount of a peptide in a samplemixture and identifying the sample from which it originated, optionallycoupled with the determination of other peptides from that sample, theprecursor protein to the degraded peptide can be identified and/orquantified with respect to the sample from which it originated. Becausethis method allows for the multiplex determination of a protein, orproteins, in more than one sample (i.e. from a sample mixture), it is amultiplex method.

In some embodiments, this invention pertains to a method comprisingreacting each of two or more samples, each sample containing one or morereactive analytes, with a different labeling reagent of a set oflabeling reagents wherein the different labeling reagents of the seteach comprise the formula: RP-X-LK-Y-RG. Consequently, one or moreanalytes of each sample are labeled with the moiety “RP-X-LK-Y-” byreaction of a nucleophile or electrophile of the analyte with theelectrophilic or nucleophilic reactive group (RG), respectively, of thedifferent labeling reagents. The labeling process can produce two ormore differentially labeled samples each comprising one or more labeledanalytes. The labeling reagents of the set can be isomeric or isobaric.The reporter of each labeling reagent can be identified with, andtherefore used to identify, the sample from which each labeled analyteoriginated.

RG is a reactive group the characteristics of which have been previouslydescribed. RP is a reporter moiety the characteristics of which havebeen previously described. The gross mass of each reporter can bedifferent for each reagent of the set. LK is a linker moiety thecharacteristics of which have been previously described. The gross massof the linker can compensate for the difference in gross mass betweenthe reporters for the different labeling reagents such that theaggregate gross mass of the reporter/linker combination is the same foreach reagent of the set. X is a bond between an atom of the reporter andan atom of the linker. Y is a bond between an atom of the linker and anatom of the reactive group (or after reaction with an analyte, Y is abond between the an atom of the linker and an atom of the analyte).Bonds X and Y fragment in at least a portion of the labeled analyteswhen subjected to dissociative energy levels in a mass spectrometer. Thecharacteristics of bonds X and Y have been previously described.

Once the analytes of each sample are labeled with the labeling reagentthat is unique to that sample, the two or more differentially labeledsamples, or a portion thereof, can be mixed to produce a sample mixture.Where quantitation is desired, the volume and/or quantity of each samplecombined to produce the sample mixture can be recorded. The volumeand/or quantity of each sample, relative to the total sample volumeand/or quantity of the sample mixture, can be used to determine theratio necessary for determining the amount (often expressed inconcentration and/or quantity) of an identified analyte in each samplefrom the analysis of the sample mixture. The sample mixture cantherefore comprise a complex mixture wherein relative amounts of thesame and/or different analytes can be identified and/or quantitated,either by relative quantitation of the amounts of analyte in each of thetwo or more samples or absolutely where a calibration standard is alsoadded to the sample mixture.

The mixture can then be subjected to spectrometry techniques wherein afirst mass analysis can be performed on the sample mixture, or fractionthereof, using a first mass analyzer. Ions of a particular mass tocharge ratio from the first mass analysis can then be selected. Theselected ions can then be subjected to dissociative energy levels (e.g.collision-induced dissociation (CID)) to thereby induce fragmentation ofthe selected ions. By subjecting the selected ions, of a particular massto charge ratio, of the labeled analytes to dissociative energy levels,both bonds X and Y can be fragmented in at least a portion of theselected ions. Fragmentation of both bonds X and Y can causefragmentation of the reporter/linker moiety as well as cause release thecharged or ionized reporter from the analyte. Ions subjected todissociative energy levels can also cause fragmentation of the analyteto thereby produce daughter fragment ions of the analyte. The ions(remaining selected ions, daughter fragment ions and charged or ionizedreporters), or a fraction thereof, can then be directed to a second massanalyzer.

In the second mass analyzer, a second mass analysis can be performed onthe selected ions, and the fragments thereof. The second mass analysiscan determine the gross mass (or m/z) and relative amount of each uniquereporter that is present at the selected mass to charge ratio as well asthe gross mass of the daughter fragment ions of at least one reactiveanalyte of the sample mixture. For each analyte present at the selectedmass to charge ratio, the daughter fragment ions can be used to identifythe analyte or analytes present at the selected mass to charge ratio.For example, this analysis can be done as previously described in thesection entitled: “Analyte Determination By Computer Assisted DatabaseAnalysis”.

In some embodiments, certain steps of the process can be repeated one ormore times. For example, in some embodiments, ions of a selected mass tocharge ratio from the first mass spectrometric analysis, different fromany previously selected mass to charge ratio, can be treated todissociative energy levels to thereby form ionized reporter moieties andionized daughter fragment ions of at least some of the selected ions, aspreviously described. A second mass analysis of the selected ions, theionized reporter moieties and the daughter fragment ions, or a fractionthereof, can be performed. The gross mass and relative amount of eachreporter moiety in the second mass analysis and the gross mass of thedaughter fragment ions can also be determined. In this way, theinformation can be made available for identifying and quantifying one ormore additional analytes from the first mass analysis.

In some embodiments, the whole process can be repeated one or moretimes. For example, it may be useful to repeat the process one or moretimes where the sample mixture has been fractionated (e.g. separated bychromatography or electrophoresis). By repeating the process on eachsample, it is possible to analyze the entire sample mixture. It iscontemplated that in some embodiments, the whole process will berepeated one or more times and within each of these repeats, certainsteps will also be repeated one or more times such as described above.In this way, the contents of sample mixture can be interrogated anddetermined to the fullest possible extent.

Those of ordinary skill in the art of mass spectrometry will appreciatethat the first and second mass analysis can be performed in a tandemmass spectrometer. Instruments suitable for performing tandem massanalysis have been previously described herein. Although tandem massspectrometers are preferred, single-stage mass spectrometers may beused. For example, analyte fragmentation may be induced by cone-voltagefragmentation, followed by mass analysis of the resulting fragmentsusing a single-stage quadrupole or time-of-flight mass spectrometer. Inother examples, analytes may be subjected to dissociative energy levelsusing a laser source and the resulting fragments recorded followingpost-source decay in time-of-flight or tandem time-of-flight (TOF-TOF)mass spectrometers.

According to the preceding disclosed multiplex methods, in someembodiments, bond X can be more or less prone to, or substantially equalto, fragmentation as compared with fragmentation of bonds of the analyte(e.g. an amide (peptide) bond in a peptide backbone). In someembodiments, bond Y can be more or less prone to fragmentation ascompared with fragmentation of bonds of the analyte (e.g. an amide(peptide) bond in a peptide backbone). In some embodiments, the linkerfor each reagent of the set is neutral in charge after the fragmentationof bonds X and Y (i.e. the linker fragments to produce a neutral loss ofmass and is therefore not observed in the MS/MS spectrum). In still someother embodiments, the position of bonds X and Y does not vary withinthe labeling reagents of a set, within the labeled analytes of a mixtureor within the labeling reagents of a kit. In yet some other embodiments,the reporter for each reagent of the set does not substantiallysub-fragment under conditions that are used to fragment the analyte(e.g. an amide (peptide) bond of a peptide backbone). In yet some otherembodiments, bond X is less prone to fragmentation as compared with bondY. In still some other embodiments, bond Y is less prone tofragmentation as compared with bond X. In still some other embodiments,bonds X and Y are of approximately the same lability or otherwise areselected such that fragmentation of one of bonds X or Y results in thefragmentation of the other of bonds X or Y. Other characteristics of thegroups that for the RP-X-LK-Y-moiety of labeled analytes have previouslybeen described.

In some embodiments, the label of each isobarically labeled analyte canbe a 5, 6 or 7 membered heterocyclic ring comprising a ring nitrogenatom that is N-alkylated with a substituted or unsubstituted acetic acidmoiety to which the analyte is linked through the carbonyl carbon of theN-alkyl acetic acid moiety, wherein each different label can compriseone or more heavy atom isotopes. The heterocyclic ring can besubstituted or unsubstituted. The heterocyclic ring can be aliphatic oraromatic. Possible substituents of the heterocylic moiety include alkyl,alkoxy and aryl groups. The substituents can comprise protected orunprotected groups, such as amine, hydroxyl or thiol groups, suitablefor linking the analyte to a support. The heterocyclic ring can compriseadditional heteroatoms such as one or more nitrogen, oxygen or sulfuratoms.

In some embodiments, labeled analytes in the sample mixture can beisobars and each comprise the general formula:

wherein: Z is O, S, NH or NR¹; each J is the same or different and is H,deuterium (D), R¹, OR¹, SR¹, NHR¹, N(R¹)₂, fluorine, chlorine, bromineor iodine; W is an atom or group that is located ortho, meta or para tothe ring nitrogen and is NH, N—R¹, N—R², P—R¹, P—R², O or S; each carbonof the heterocyclic ring has the formula CJ₂; each R¹ is the same ordifferent and is an alkyl group comprising one to eight carbon atomswhich may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms; and R² is an amino alkyl, hydroxy alkyl, thio alkyl group or acleavable linker that cleavably links the reagent to a solid supportwherein the amino alkyl, hydroxy alkyl or thio alkyl group comprises oneto eight carbon atoms, which may optionally contain a heteroatom or asubstituted or unsubstituted aryl group, and wherein the carbon atoms ofthe alkyl and aryl groups independently comprise linked hydrogen,deuterium and/or fluorine atoms.

For example, the sample mixture can comprise one or more isobaricallylabeled analytes of the general formula:

wherein isotopes of carbon 13 and oxygen 18 are used to balance thegross mass between the morpholine reporter and the carbonyl linker ofthe different labeling reagents.

Morpholine labeling reagents suitable to produce labeled analytes ofthis general structure can be prepared by numerous synthetic routes. Forexample, isotopically labeled or non-isotopically morpholine compoundscan be reacted with isotopically labeled or non-isotopically labeledbromoacetic acid compounds as described in Example 1. It should likewisebe apparent that a ring-substituted morpholine and/or substitutedbromoacetic acid starting materials can also be selected and used by oneof skill in the art without the exercise of undue experimentation (withlittle or no change to the above described procedure or other procedureswell-known in the art) to thereby produce various different morpholinebased labeling reagents, of differing heavy atom isotope content (i.e.isotopically coded), that can be used in the sets or kits of thisinvention.

Instead of morpholine, it is possible to choose a substituted orunsubstituted piperidine of desired isotopic distribution. Whenpiperidine is chosen, the isotopes D (deuterium) ¹³C or ¹⁵N can besubstituted for H, ¹²C and ¹⁴N, respectively, and used to alter thegross mass of the reagents of a set of labeling reagents in a mannersimilar to that illustrated for morpholine except that in the case ofpiperidine, ¹⁸O is not used in the ring atoms. An exemplary synthesis ofa piperidine, optionally using isotopically enriched starting materials,is described in Example 6.

The sample mixture can comprise one or more isobarically labeledanalytes of the formula:

wherein isotopes of carbon 13, oxygen 18 and nitrogen 15 are used tobalance the gross mass between the reporter and the carbonyl linker ofthe different labeling reagents. Piperazine labeling reagents suitableto produce labeled analytes of this general structure can be prepared bynumerous synthetic routes. For example, heavy or light piperazinecompounds can be reacted with heavy or light labeled bromoacetic acidcompounds as described in Example 7. With reference to FIGS. 9A and 9B,a general schematic is shown for two different synthetic routes toisotopically enriched piperazines using readily available heavy or lightstarting materials.

Specifically with reference to FIG. 9A, two equivalents of ¹⁵N-labeledglycine 1 can be condensed to form the bis-isotopically labeleddi-ketopiperazine 2 (the isotopic label is represented by the * in theFigure). The di-ketopiperazine can then be reduced to an isotopicallylabeled piperazine. The isotopically labeled piperazine can then bereacted with bromoacetic acid and converted to an active ester 3 asdescribed in Example 7.

Specifically with reference to FIG. 9B, bis-¹⁵N-labeled ethylenediamine4 can be condensed with oxalic acid 5 to for the bis-isotopicallylabeled di-ketopiperazine 6 (the isotopic label is represented by the *in the Figure). The di-ketopiperazine can then be reduced to anisotopically labeled piperazine. The isotopically labeled piperazine canthen be reacted with bromoacetic acid and converted to an active ester 3as described in Example 7.

It should likewise be apparent that a ring-substituted piperazine can bemade using the above-described methods by merely choosing appropriatelysubstituted starting materials. Where appropriate, a substitutedbromoacetic acid (either heavy or light) can likewise be used. By heavywe mean that the compound is isotopically enriched with one or moreheave atom isotopes). By light we mean that it is not isotopicallyenriched. Accordingly, appropriately substituted starting materials canbe selected to thereby produce various different piperazine basedlabeling reagents that can be used in the sets of this invention.

For example, the sample mixture can comprise one or more isobaricallylabeled analytes of the formula:

wherein: isotopes of carbon 13, oxygen 18 and nitrogen 15 are used tobalance the gross mass between the reporter and the carbonyl linker ofthe different labeling reagents and wherein; 1) each R¹ is the same ordifferent and is an alkyl group comprising one to eight carbon atomswhich may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms; and 2) each K is independently selected as hydrogen or an aminoacid side chain. Substituted piperazine labeling reagents suitable toproduce labeled analytes of this general structure can be prepared bynumerous synthetic routes.

For example, with reference to FIG. 10, N-alkyl substituted piperazinereagents can be prepared in accordance with the illustrated procedure.The tert-butyloxycarbonyl (t-boc) protected glycine 10 can be condensedwith the ester (e.g. ethyl ester) of N-methyl-glycine 11 to thereby formthe ester of the t-boc protected glycine-N-methyl-glycine dimer 12. Thegly-gly dimer 12 can then be cyclized by removal of the t-boc protectinggroup followed by condensation to thereby form the acid salt of theN-methyl-di-ketopiperazine 13. The acid salt of 13 can be neutralizedand reduced to form the N-methyl-piperazine 14. The N-methyl-piperazine14 can then be reacted with bromoacetic acid 15 (or substituted versionsthereof) and converted to an active ester 16 as described in Example 7.

It should be apparent that a ring-substituted piperazine can be madeusing the above-described method by merely choosing an amino acid orN-methyl amino acid (or ester thereof) other than glycine (e.g. alanine,phenylalanine, leucine, isoleucine, valine, asparagine, apartic acid,etc). It should likewise be apparent that the amino acids can beisotopically labeled in a manner suitable for preparing ring substitutedpiperazines having the desired distribution of isotopes necessary toprepare sets of isobaric labeling reagents.

N-alkyl substituted piperazine reagents can be prepared in accordance bystill another illustrated procedure. With reference to FIG. 11, glycinemethyl ester 21 can be reacted with the ethyl ester of bromoacetic acid22 to form the diethyl iminodiacetate 23. The diester of the diethyliminodiacetate 23 can be converted to a di-acid chloride 24 by treatmentan appropriate reagent (e.g. thionyl chloride). The di-acid chloride 24can then be reacted with, for example, an alkyl amine (e.g. methylamine) to form an N-alkyl-di-ketopiperazine 25. TheN-alkyl-di-ketopiperazine 25 can then be reduced to form theN-alkyl-piperazine 26. The N-alkyl-piperazine can then be reacted withbromoacetic acid and converted to an active ester 27 as described inExample 7.

It should be apparent that a ring-substituted piperazine can be madeusing the above-described method by merely choosing an ester of an aminoacid other than glycine (e.g. alanine, phenylalanine, leucine,isoleucine, valine, asparagine, apartic acid, etc) or a substitutedversion of bromoacetic acid. It should likewise be apparent that theamino acids and bromoacetic acid (and its substituted derivatives) canbe isotopically labeled in a manner suitable for preparing ringsubstituted piperazines having the desired distribution of isotopesnecessary to prepare sets of isobaric labeling reagents. It should befurther apparent that choosing an alkyl diamine, hydroxyalkyl amine orthioalkylamine, or isotopically labeled version thereof, instead of analkyl amine can be used to produce the support bound labeling reagentsas described in more detail below.

In yet some other embodiments of the method, labeled analytes in thesample mixture are isobars and each comprise the formula:

wherein: Z is O, S, NH or NR¹; each J is the same or different and isselected from the group consisting of: H, deuterium (D), R¹, OR¹, SR¹,NHR¹, N(R¹)₂, fluorine, chlorine, bromine and iodine; each R¹ is thesame or different and is an alkyl group comprising one to eight carbonatoms which may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms.

For example, the sample mixture can comprise two or more isobaricallylabeled analytes of the formula:

wherein isotopes of carbon 13 and oxygen 18 are used to balance thegross mass between the reporter and the carbonyl linker of the differentlabeling reagents. Substituted labeling reagents suitable to producelabeled analytes of this general structure can be prepared by thegeneral process described in Example 8.

In still some other embodiments of this invention, each differentlabeling reagent of a set or kit of labeling reagents can be linked to asupport through a cleavable linker such that each different sample canbe reacted with a support carrying a different labeling reagent. In someembodiments, the supports can themselves be used for the labeling ofreactive analytes. In some embodiments, the labeling reagents can beremoved from the supports and then used, in some cases after subsequentprocessing (e.g. protection of reactive groups), for the labeling ofreactive analytes.

According to some embodiments, the analytes from a sample can be reactedwith the solid support (each sample being reacted with a different solidsupport and therefore a different reporter) and the resin boundcomponents of the sample that do not react with the reactive group canbe optionally washed away. The labeled analyte or analytes can then beremoved from each solid support by treating the support under conditionsthat cleave the cleavable linker and thereby release thereporter/linker/analyte complex from the support. Each support can besimilarly treated under conditions that cleave the cleavable linker tothereby obtain two or more different samples, each sample comprising oneor more labeled analytes wherein the labeled analytes associated with aparticular sample can be identified and/or quantified by the uniquereporter linked thereto. The collected samples can then be mixed to forma sample mixture, as previously described.

For example, each different labeling reagent of the set used in thepreviously described method can be a solid support of the formula:E-F-RP-X-LK-Y-RG, wherein; RG, X, Y, RP and LK have been describedpreviously. E is a solid support and F is a cleavable linker linked tothe solid support and cleavably linked to the reporter. Supports of thisgeneral formula can be prepared as described in Example 9.

In some embodiments, a set of support bound labeling reagents can bebased on labeled N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine derivatives. Both heavy and light piperazinederivatives can be prepared. The labeled N-(aminoalkyl), N-(thioalkyl)or N-(hydroxyalkyl)-piperazine derivatives can be formed, for example,by using the procedure illustrated in FIG. 11 starting with an alkyldiamine, thioalkyl amine or hydroxyalkyl amine as the N-alkyl amine (seethe discussion of FIG. 11, above). The alkyl diamine, thioalkyl amine orhydroxyalkyl amine can be heavy or light where appropriate for synthesisof a desired N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine derivative. The amino, hydroxyl or thiolgroup of the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine derivatives can be protected as appropriate.When an alkyl diamine, thioalkylamine or hydroxyalkyl amine is used, thepiperazine can comprise an N-aminoalkyl, N-thioalkyl or N-hydroxyalkylmoiety wherein the amino, hydroxyl or thiol group of the moiety can bereacted with the cleavable linker on a support to thereby cleavably linkthe piperazine, prepared from the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine derivative, to the support.

The support comprising a labeling reagent can be prepared by any ofseveral methods. In some embodiments, the amino, hydroxyl or thiol groupof the N-(aminoalkyl), N-(thioalkyl) or N-(hydroxyalkyl)-piperazine canbe reacted with the cleavable linker of a suitable support. Thecleavable linker can be a “sterically hindered cleavable linker” (See:Example 9). The piperazine can be reacted with isotopically labeled ornon-isotopically labeled haloacetic acid (substituted or unsubstituted)depending on the nature of the labeling reagent desired for the set oflabeling reagents. Thereafter the carboxylic acid can be converted to anactive ester. The active ester can be reacted with analytes of a sampleto thereby label the analytes with the labeling reagent of the support.Cleavage of the cleavable linker will release the labeled analyte fromthe support. This process can be repeated with an unique piperazinebased labeling reagent for the preparation of the different supports ofa set of labeling supports.

In some embodiments, the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine can be first reacted with isotopicallylabeled or non-isotopically labeled haloacetic acid (substituted orunsubstituted), or an ester thereof. Preferably, the amino, hydroxyl orthiol group of the N-(aminoalkyl), N-(thioalkyl) orN-(hydroxyalkyl)-piperazine can be protected with a suitable protectingreagent (For a list of suitable protecting groups See: Green et al.,Protecting Groups In Organic Synthesis, Third Edition, John Wiley &Sons, Inc. New York, 1999). The unprotected amino, thiol or hydroxylgroup of the resulting bis-alkylated piperazine can then be reacted withthe cleavable linker of a suitable support. Thereafter the carboxylicacid can be converted to an active ester. If the haloacetic acidcompound was an ester, the ester can be saponified prior to conversionto an active ester. The active ester can be reacted with analytes of asample to thereby label the analytes with the labeling reagent of thesupport. Cleavage of the cleavable linker will release the labeledanalyte from the support. This process can be repeated with a uniquepiperazine based labeling reagent for the preparation of the differentsupports of a set of labeling supports.

Therefore, in some embodiments, the set of labeling reagents cancomprise one or more of the following support bound labeling reagents:

wherein RG, E and F have been previously described. According to themethod, G can be an amino alkyl, hydroxy alkyl or thio alkyl group,cleavably linked to the cleavable linker wherein the amino alkyl,hydroxy alkyl or thio alkyl group comprises one to eight carbon atoms,which may optionally contain a heteroatom or a substituted orunsubstituted aryl group, and wherein the carbon atoms of the alkyl andaryl groups independently comprise linked hydrogen, deuterium and/orfluorine atoms. Each carbon of the heterocyclic ring can have theformula CJ₂, wherein each J is the same or different and is selectedfrom the group consisting of H, deuterium (D), R¹, OR¹, SR¹, NHR¹,N(R¹)₂, fluorine, chlorine, bromine and iodine. Each R¹ can be the sameor different and is an alkyl group comprising one to eight carbon atomswhich may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms.

In some embodiments, the labeled analytes can be generated by firstreacting the analyte with a support comprising the labeling reagent,cleavably linked to the support through a cleavable linker, and thencleaving the labeled analyte from the support. Accordingly, a samplemixture can comprise one or more isobarically labeled analytes of theformula:

wherein: G′ can be an amino alkyl, hydroxy alkyl or thio alkyl groupcomprising one to eight carbon atoms which may optionally contain aheteroatom or a substituted or unsubstituted aryl group wherein thecarbon atoms of the alkyl and aryl groups independently comprise linkedhydrogen and/or deuterium atoms. Each carbon of the heterocyclic ringcan have the formula CJ₂, wherein each J is the same or different and isselected from the group consisting of: H, deuterium (D), R¹, OR¹, SR¹,NHR¹, N(R¹)₂, fluorine, chlorine, bromine and iodine. Each R¹ can be thesame or different and is an alkyl group comprising one to eight carbonatoms which may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms. Here the alkyl amine group, hydroxy alkyl group or thio alkylgroup can be the moiety that was linked to the cleavable linker of thesolid support. The product of each cleavage reaction can be combined toproduce a sample mixture suitable for analysis of labeled analytes bythe methods described herein.

In some embodiments, methods of the invention can further comprisedigesting each sample with at least one enzyme to partially, or fully,degrade components of the sample prior to performing the labeling of theanalytes of the sample (Also see the above section entitled: “SampleProcessing”). For example, the enzyme can be a protease (to degradeproteins and peptides) or a nuclease (to degrade nucleic acids). Theenzymes may also be used together to thereby degrade sample components.The enzyme can be a proteolytic enzyme such as trypsin, papain, pepsin,ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin or carboxypeptidaseC.

In some embodiments, methods can further comprise separating the samplemixture prior to performing the first mass analysis (Also see the abovesection entitled: “Separation Of The Sample Mixture”). In this mannerthe first mass analysis can be performed on only a fraction of thesample mixture. The separation can be performed by any separationsmethod, including by chromatography or by electrophoresis. For example,liquid chromatography/mass spectrometry (LC/MS) can be used to effectsuch a sample separation and mass analysis. Moreover, anychromatographic separation process suitable to separate the analytes ofinterest can be used. Non-limiting examples of suitable chromatographicand electrophoretic separations processes have been described herein.

In still other embodiments, the methods of the invention can compriseboth an enzyme treatment to degrade sample components and a separationsstep.

As described previously, it is possible to determine the analyteassociated with the selected ions by analysis of the gross mass of thedaughter fragment ions. One such method of determination is described inthe section entitled: “Analyte Determination By Computer AssistedDatabase Analysis”.

Once the analyte has been determined, information regarding the grossmass and relative amount of each reporter moiety in the second massanalysis and the gross mass of daughter fragment ions provides the basisto determine other information about the sample mixture. The amount ofreporter can be determined by peak intensity in the mass spectrum. Insome embodiments, the amount of reporter can be determined by analysisof the peak height or peak width of the reporter (signature ion) signalobtained using the mass spectrometer. Because each sample can be labeledwith a different labeling reagent and each labeling reagent can comprisea unique reporter that can be correlated with a particular sample,determination of the different reporters in the second mass analysisidentifies the sample from which the ions of the selected analyteoriginated. Where multiple reporters are found (e.g. according to themultiplex methods of the invention), the relative amount of eachreporter can be determined with respect to the other reporters. Becausethe relative amount of each reporter determined in the second massanalysis correlates with the relative amount of an analyte in the samplemixture, the relative amount (often expressed as concentration and/orquantity) of the analyte in each sample combined to form the samplemixture can be determined. As appropriate, a correction of peakintensity associated with the reporters can be performed for naturallyoccurring, or artificially created, isotopic abundance, as previouslydiscussed. More specifically, where the volume and/or quantity of eachsample that is combined to the sample mixture is known, the relativeamount (often expressed as concentration and/or quantity) of the analytein each sample can be calculated based upon the relative amount of eachreporter determined in the second mass analysis.

This analysis can be repeated one or more times on selected ions of adifferent mass to charge ratio to thereby obtain the relative amount ofone or more additional analytes in each sample combined to form thesample mixture. As appropriate, a correction of peak intensityassociated with the reporters can be performed for naturally occurring,or artificially created, isotopic abundance.

Alternatively, where a calibration standard comprising a unique reporterlinked to an analyte, having the selected mass to charge ratio, has beenadded to the sample mixture in a known amount (often expressed as aconcentration and/or quantity), the amount of the unique reporterassociated with the calibration standard can be used to determine theabsolute amount (often expressed as a concentration and/or quantity) ofthe analyte in each of the samples combined to form the sample mixture.This is possible because the amount of analyte associated with thereporter for the calibration standard is known and the relative amountsof all other reporters can be determined for the labeled analyteassociated with the selected ions. Since the relative amount ofreporter, determined for each of the unique reporters (including thereporter for the calibration standard), is proportional to the amount ofthe analyte associated with each sample combined to form the samplemixture, the absolute amount (often expressed as a concentration and/orquantity) of the analyte in each of the samples can be determined basedupon a ratio calculated with respect to the formulation used to producethe sample mixture. As appropriate, a correction of peak intensityassociated with the reporters can be performed for naturally occurring,or artificially created, isotopic abundance.

This analysis can be repeated one or more times on selected ions of adifferent mass to charge ratio to thereby obtain the absolute amount ofone or more additional analytes in each sample combined to form thesample mixture. As appropriate, a correction of peak intensityassociated with the reporters can be performed for naturally occurring,or artificially created, isotopic abundance.

In some embodiments, the methods can be practiced with digestion and/orseparation steps. In some embodiments, the steps of the methods, with orwithout the digestion and/or separation steps, can be repeated one ormore times to thereby identify and/or quantify one or more otheranalytes in a sample or one or more analytes in each of the two or moresamples (including samples labeled with support bound labelingreagents). Depending of whether or not a calibration standard is presentin the sample mixture for a particular analyte, the quantitation can berelative to the other labeled analytes, or it can be absolute. Such ananalysis method can be particularly useful for proteomic analysis ofmultiplex samples of a complex nature, especially where a preliminaryseparation of the labeled analytes (e.g. liquid chromatography orelectrophoretic separation) precedes the first mass analysis.

In some embodiments, the analytes can be peptides in a sample or samplemixture. Analysis of the peptides in a sample, or sample mixture, can beused to determine the amount (often expressed as a concentration and/orquantity) of identifiable proteins in the sample or sample mixturewherein proteins in one or more samples can be degraded prior to thefirst mass analysis. Moreover, the information from different samplescan be compared for the purpose of making determinations, such as forthe comparison of the effect on the amount of the protein in cells thatare incubated with differing concentrations of a substance that mayaffect cell growth. Other, non-limiting examples may include comparisonof the expressed protein components of diseased and healthy tissue orcell cultures. This may encompass comparison of expressed protein levelsin cells, tissues or biological fluids following infection with aninfective agent such as a bacteria or virus or other disease states suchas cancer. In other examples, changes in protein concentration over time(time-course) studies may be undertaken to examine the effect of drugtreatment on the expressed protein component of cells or tissues. Instill other examples, the information from different samples taken overtime may be used to detect and monitor the concentration of specificproteins in tissues, organs or biological fluids as a result of disease(e.g. cancer) or infection.

In some embodiments, the analyte can be a nucleic acid fragment in asample or sample mixture. The information on the nucleic acid fragmentscan be used to determine the amount (often expressed as a concentrationand/or quantity) of identifiable nucleic acid molecules in the sample orsample mixture wherein the sample was degraded prior to the first massanalysis. Moreover, the information from the different samples can becompared for the purpose of making determinations as described above.

B. Mixtures

In some embodiments, this invention pertains to mixtures (i.e. samplemixtures). The mixtures can comprise at least two differentially labeledanalytes, wherein each of the two-labeled analytes can originate from adifferent sample and comprise the formula: RP-X-LK-Y-Analyte. For eachdifferent label, some of the labeled analytes of the mixture can be thesame and some of the labeled analytes can be different. The atoms,moieties or bonds, X, Y, RP and LK have been previously described andtheir characteristics disclosed. The mixture can be formed by mixingall, or a part, of the product of two or more labeling reactions whereineach labeling reaction uses a different labeling reagent of the generalformula: RP-X-LK-Y-RG, wherein atoms, moieties or bonds X, Y, RP, LK RGhave been previously described and their characteristics disclosed. Thelabeling reagents can be isotopically coded isomeric or isobariclabeling reagents. The unique reporter of each different labelingreagent can indicate from which labeling reaction each of the two ormore labeled analytes is derived. The labeling reagents can be isomericor isobaric. Hence, two or more of the labeled analytes of a mixture canbe isomeric or isobaric. The mixture can be the sample mixture asdisclosed in any of the above-described methods. Characteristics of thelabeling reagents and labeled analytes associated with those methodshave been previously discussed.

The analytes of the mixture can be peptides. The analytes of the mixturecan be proteins. The analytes of the mixture can be peptides andproteins. The analytes of the mixture can be nucleic acid molecules. Theanalytes of the mixture can be carbohydrates. The analytes of themixture can be lipids. The analytes of the mixture can be steroids. Theanalytes of the mixture can be small molecules of less than 1500daltons. The analytes of the mixture comprise two or more analyte types.The analyte types can, for example, be selected from peptides, proteins,nucleic acids carbohydrates, lipids, steroids and/or small molecules ofless than 1500 daltons.

In some embodiments, the label of each isobarically labeled analyte canbe a 5, 6 or 7 membered heterocyclic ring comprising a ring nitrogenatom that is N-alkylated with a substituted or unsubstituted acetic acidmoiety to which the analyte is linked through the carbonyl carbon of theN-alkyl acetic acid moiety, wherein each different label comprises oneor more heavy atom isotopes. The heterocyclic ring can be substituted orunsubstituted. The heterocyclic ring can be aliphatic or aromatic.Possible substituents of the heterocylic moiety include alkyl, alkoxyand aryl groups. The substituents can comprise protected or unprotectedgroups, such as amine, hydroxyl or thiol groups, suitable for lining theanalyte to a support. The heterocyclic ring can comprise additionalheteroatoms such as one or more nitrogen, oxygen or sulfur atoms.

In some embodiments, the labeled analytes of the mixture are isobars andeach comprise the formula:

wherein Z, J and W have been previously described and theircharacteristics disclosed. For example, the sample mixture can compriseone or more isobarically labeled analytes of the formula:

wherein isotopes of carbon 13 and oxygen 18 are used to balance thegross mass between the morpholine reporter and the carbonyl linker ofthe different labeling reagents.

In some embodiments, the sample mixture can comprise one or moreisobarically labeled analytes of the formula:

wherein isotopes of carbon 13, oxygen 18 and nitrogen 15 are used tobalance the gross mass between the reporter and the carbonyl linker ofthe different labeling reagents. In some embodiments, the sample mixturecan comprise one or more isobarically labeled analytes of the formula:

wherein: isotopes of carbon 13, oxygen 18 and nitrogen 15 are used tobalance the gross mass between the reporter and the carbonyl linker ofthe different labeling reagents and wherein; 1) each R¹ is the same ordifferent and is an alkyl group comprising one to eight carbon atomswhich may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms; and 2) each K is independently selected as hydrogen or an aminoacid side chain.

In some embodiments, the labeled analytes of the mixture are isobars andeach comprise the formula:

wherein: Z, J and R′ have been previously described and theircharacteristics disclosed. For example, the sample mixture can compriseone or more isobarically labeled analytes of the formula:

wherein isotopes of carbon 13 and oxygen 18 are used to balance thegross mass between the reporter and the carbonyl linker of the differentlabeling reagents.

In other embodiments, the labeled analytes can be generated by firstreacting the analyte with a support comprising the labeling reagent,cleavably linked to the support through a cleavable linker, and thencleaving the labeled analyte from the support. For example the labeledanalytes of the mixture can be one or more isobars comprising thegeneral formula:

wherein: G′ has been previously described and its characteristicsdisclosed.

C. Kits

In some embodiments, this invention pertains to kits. The kits cancomprise a set of two or more labeling reagents of the formula:RP-X-LK-Y-RG and one or more reagents, containers, enzymes, buffersand/or instructions. The atoms, moieties or bonds X, Y, RP, LK RG havebeen previously described and their characteristics disclosed. Thelabeling reagents of a kit can be isomeric or isobaric. Other propertiesof the labeling reagents of the kits have likewise been disclosed. Forexample, the kits can be useful for the multiplex analysis of one ormore analytes in the same sample, or in two or more different samples.

In some embodiments, the label of each isobarically labeled analyte canbe a 5, 6 or 7 membered heterocyclic ring comprising a ring nitrogenatom that is N-alkylated with a substituted or unsubstituted acetic acidmoiety to which the analyte is linked through the carbonyl carbon of theN-alkyl acetic acid moiety, wherein each different label comprises oneor more heavy atom isotopes. The heterocyclic ring can be substituted orunsubstituted. The heterocyclic ring can be aliphatic or aromatic.Possible substituents of the heterocylic moiety include alkyl, alkoxyand aryl groups. The substituents can comprise protected or unprotectedgroups, such as amine, hydroxyl or thiol groups, suitable for linkingthe analyte to a support. The heterocyclic ring can comprise additionalheteroatoms such as one or more nitrogen, oxygen or sulfur atoms.

In some embodiments, the different reagents of a kit are isobars andeach comprise the formula:

wherein RG, Z, J and W have been previously described and theircharacteristics disclosed. For example, the reagents of a kit cancomprise one or more isobarically labeled reagents of the formula:

wherein RG is the reactive group and isotopes of carbon 13 and oxygen 18are used to balance the gross mass between the morpholine reporter andthe carbonyl linker of the different labeling reagents.

In some embodiments, the kit can comprise one or more isobaricallylabeled reagents of the formula:

wherein RG is the reactive group and isotopes of carbon 13, oxygen 18and nitrogen 15 are used to balance the gross mass between the reporterand the carbonyl linker of the different labeling reagents. In someembodiments, the reagents of a kit can comprise one or more isobaricallylabeled reagents of the formula:

wherein: isotopes of carbon 13, oxygen 18 and nitrogen 15 are used tobalance the gross mass between the reporter and the carbonyl linker ofthe different labeling reagents and wherein; 1) each R¹ is the same ordifferent and is an alkyl group comprising one to eight carbon atomswhich may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen, deuterium and/or fluorineatoms; and 2) each K is independently selected as hydrogen or an aminoacid side chain. In yet other embodiments, the labeled analytes of thekit are isobars and each comprises the formula:

wherein: RG Z, J and R′ have been previously described and theircharacteristics disclosed. For example, the reagents of a kit cancomprise one or more isobarically labeled analytes of the formula:

wherein RG has been previously described and disclosed and isotopes ofcarbon 13 and oxygen 18 are used to balance the gross mass between thereporter and the carbonyl linker of the different labeling reagents.

In some embodiments, this invention pertains to kits comprising one ormore sets of supports, each support comprising a different labelingreagent, cleavably linked to the support through a cleavable linker. Forexample, the cleavable linker can be chemically or photolyticallycleavable. The supports can be reacted with different samples therebylabeling the analytes of a sample with the same reporter/linker, andanalytes of different samples with different reporter/linkercombinations. Supports of a set that can be used in embodiments of thisinvention have the general formula: E-F-G-RP-X-LK-Y-RG, wherein E, F, G,RP, X, LK, Y and RG have been previously defined herein and theircharacteristics disclosed. Each different support of the set cancomprise a unique reporter.

For example the supports of a kit can comprise two or more of thereagent supports of the formula:

wherein: E, F, G and RG have been previously described and theircharacteristics disclosed.

In some embodiments, the kit comprises a proteolytic enzyme. Theproteolytic enzyme can be trypsin, papain, pepsin, ArgC, LysC, V8protease, AspN, pronase, chymotrypsin or carboxypeptidase C. In someembodiments, the kit can comprise instructions for using the labelingreagents to differentially label the analytes of different samples.

D. Compositions

In some embodiments, this invention pertains to compositions that can beused as labeling reagents. The compositions can be labeling reagents ofthe formula: RP-X-LK-Y-RG, wherein the atoms, moieties or bonds X, Y,RP, LK RG have been previously described and their characteristicsdisclosed. The labeling reagents can be isomeric or isobaric. Otherproperties of the labeling reagents have likewise been disclosed. Forexample, the labeling reagents can be useful for the multiplex analysisof one or more analytes in the same sample, or in two or more differentsamples.

The labeling reagents can be isotopically enriched (coded) with at leastone heavy atom isotope. The labeling reagents can be isotopicallyenriched to comprise two or more heavy atom isotopes. The labelingreagents can be isotopically enriched to comprise three or more heavyatom isotopes. The labeling reagents can be isotopically enriched tocomprise four or more heavy atom isotopes. In some embodiments, at leastone heavy atom isotope is incorporated into a carbonyl or thiocarbonylgroup of the labeling reagent and at least one other heavy atom isotopeis incorporated into the reporter group of the labeling reagent.

Each incorporated heavy atom isotope can be present in at least 80percent isotopic purity. Each incorporated heavy atom isotope can bepresent in at least 93 percent isotopic purity. Each incorporated heavyatom isotope can be present in at least 96 percent isotopic purity.

The labeling reagents comprise a reporter group that contains a fixedcharge or that is ionizable. The reporter group therefore can includebasic or acidic moieties that are easily ionized. In some embodiments,the reporter can be a morpholine, piperidine or piperazine compound. Insome embodiments, the reporter can be a carboxylic acid, sulfonic acidor phosphoric acid group containing compound. Accordingly, is someembodiments, the labeling reagents can be isolated in their salt form.For example, piperazine containing labeling reagents can be obtained asa mono-TFA salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt. Thenumber of counterions present in the labeling reagent can depend in thenumber of acidic and/or basic groups present in the labeling reagent.

In some embodiments, the labeling reagents can comprise a carbonyl orthiocarbonyl linker. Labeling reagents comprising a carbonyl orthiocarbonyl linker can be used in active ester form for the labeling ofanalytes. In an active ester, an alcohol group forms a leaving group(LG). In some embodiments, the alcohol (LG) of the active ester can havethe formula:

wherein X is O or S. The active ester can be an N-hydroxysuccinimidylester.

In some embodiments, the active ester compound can be a 5, 6 or 7membered heterocyclic ring comprising a ring nitrogen atom that isN-alkylated with a substituted or unsubstituted acetic acid moiety towhich the alcohol moiety of the active ester is linked through thecarbonyl carbon of the N-alkyl acetic acid moiety, wherein the compoundis isotopically enriched with one or more heavy atom isotopes. Theheterocyclic ring of the active ester can be substituted with one ormore substituents. The one or more substituents can be alkyl, alkoxy oraryl groups. The one or more substituents can be alkylamine,alkylhydroxy or alkylthio groups. The one or more substituents can beprotected or unprotected amine groups, hydroxyl groups or thiol groups.The heterocyclic ring can be aliphatic. The heterocyclic ring can bearomatic. The heterocyclic ring can comprise one or more additionalnitrogen, oxygen or sulfur atoms.

In some embodiments, the active ester compound can be an N-substitutedmorpholine acetic acid active ester compound of the formula:

or a salt thereof, wherein; LG is the leaving group of an active ester;X is O or S; each Z is independently hydrogen, deuterium, fluorine,chlorine, bromine, iodine, an amino acid side chain or a straight chainor branched C1-C6 alkyl group that may optionally contain a substitutedor unsubstituted aryl group wherein the carbon atoms of the alkyl andaryl groups each independently comprise linked hydrogen, deuterium orfluorine atoms. In some embodiments, Z independently can be hydrogen,deuterium, fluorine, chlorine, bromine or iodine. In some embodiments, Zindependently can be hydrogen, methyl or methoxy. In some embodiments, Xis ¹⁶O or ¹⁸O. The nitrogen atom of the morpholine ring can be ¹⁴N or¹⁵N. In some embodiments, the active ester is a compound comprising theformula:

wherein each C* is independently ¹²C or ¹³C; LG is the leaving group ofan active ester; X is O or S; and each Z is independently hydrogen,deuterium, fluorine, chlorine, bromine, iodine, an amino acid side chainor a straight chain or branched C1-C6 alkyl group that may optionallycontain a substituted or unsubstituted aryl group wherein the carbonatoms of the alkyl and aryl groups each independently comprise linkedhydrogen, deuterium or fluorine atoms.

In some embodiments, the active ester compound can be an N-substitutedpiperidine acetic acid active ester compound of the formula:

or a salt thereof, wherein; LG is the leaving group of an active ester;X is O or S; each Z is independently hydrogen, deuterium, fluorine,chlorine, bromine, iodine, an amino acid side chain or a straight chainor branched C1-C6 alkyl group that may optionally contain a substitutedor unsubstituted aryl group wherein the carbon atoms of the alkyl andaryl groups each independently comprise linked hydrogen, deuterium orfluorine atoms. In some embodiments, Z independently can be hydrogen,deuterium, fluorine, chlorine, bromine or iodine. In some embodiments, Zindependently can be hydrogen, methyl or methoxy. In some embodiments Xis ¹⁶O or ¹⁸O. The nitrogen atom of the piperidine ring can be ¹⁴N or¹⁵N. In some embodiments, the active ester is a compound comprising theformula:

wherein each C* is independently ¹²C or ¹³C; LG is the leaving group ofan active ester; X is O or S; and each Z is independently hydrogen,deuterium, fluorine, chlorine, bromine, iodine, an amino acid side chainor a straight chain or branched C1-C6 alkyl group that may optionallycontain a substituted or unsubstituted aryl group wherein the carbonatoms of the alkyl and aryl groups each independently comprise linkedhydrogen, deuterium or fluorine atoms.

In some embodiments, the active ester compound can be an N-substitutedpiperidine acetic acid active ester compound of the formula:

or a salt thereof, wherein; LG is the leaving group of an active ester;X is O or S; Pg is an amine protecting group; and each Z isindependently hydrogen, deuterium, fluorine, chlorine, bromine, iodine,an amino acid side chain or a straight chain or branched C1-C6 alkylgroup that may optionally contain a substituted or unsubstituted arylgroup wherein the carbon atoms of the alkyl and aryl groups eachindependently comprise linked hydrogen, deuterium or fluorine atoms. Insome embodiments, Z independently can be hydrogen, deuterium, fluorine,chlorine, bromine or iodine. In some embodiments, Z independently can behydrogen, methyl or methoxy. In some embodiments X is ¹⁶O or ¹⁸O. Insome embodiments, each nitrogen atom of the piperazine ring is ¹⁴N or¹⁵N. In some embodiments, the active ester is a compound comprising theformula:

wherein each C* is independently ¹²C or ¹³C; LG is the leaving group ofan active ester; X is O or S; Pg is an amine protecting group and each Zis independently hydrogen, deuterium, fluorine, chlorine, bromine,iodine, an amino acid side chain or a straight chain or branched C1-C6alkyl group that may optionally contain a substituted or unsubstitutedaryl group wherein the carbon atoms of the alkyl and aryl groups eachindependently comprise linked hydrogen, deuterium or fluorine atoms.

Having described embodiments of the invention, it will now becomeapparent to one of skill in the art that other embodiments incorporatingthe concepts may be used. It is felt, therefore, that these embodimentsshould not be limited to disclosed embodiments but rather should belimited only by the spirit and scope of the invention.

EXAMPLES

This invention is now illustrated by the following examples that are notintended to be limiting in any way.

Example 1 Synthesis of Morpholine Acetic Acid

Bromoacetic acid (2 g, 14.4 mole) was dissolved in tetrahydrofuran (50mL) and added dropwise to a stirred solution of morpholine (3.76 g, 43.2mole) in tetrahydrofuran (THF, 20 mL). The solution was stirred at roomtemperature for three days. The white solid (4.17 g) was filtered,washed with THF (100 mL), and recrystallised from hot ethanol (EtOH),Yield: 2.59 g: IR:1740 cm-1. For the two different isobaric versions ofmorpholine acetic acid, either bromoacetic-1-¹³C acid (Aldrich PN27,933-1) or bromoacetic-2-¹³C acid (Aldrich PN 27,935-8) wassubstituted for bromoacetic acid.

Example 2 Synthesis of Morpholine Acetic Acid N-Hydroxysuccinimide Ester

Dimethylformamide (dry, 1.75 g, 0.024M) was dissolved in tetrahydrofuran(dry, 30 mLs). This solution was added dropwise to a stirred solution ofthionyl chloride (2.85 g, 0.024M) dissolved in tetrahydrofuran (dry, 20mLs) and cooled in an ice bath. After complete addition and 30 minuteson ice, the ice bath was removed and solid N-hydroxysuccinimide (2 g,0.017 M) was added (which completely dissolved) immediately followed bysolid pre-powdered morpholine acetic acid [or −1-¹³C or −2-¹³Cmorpholine acetic acid] (3.64 g, 0.016M). The morpholine acetic aciddissolved slowly giving a homogeneous solution that rapidly becamecloudy. The reaction was left vigorously stirring over night at roomtemperature. The white solid was washed with tetrahydrofuran and driedunder vacuum, weight 3.65 g (67%), IR spectrum 1828.0 cm-1, 1790.0 cm-1,1736.0 cm-1.

Example 3 Analyte Determination and Relative Quantitation in Two Samples

100 pmole amounts of freeze-dried Glu-Fibrinopeptide B (Sigma) werereacted with 200 μl of freshly-made 2% w/v solutions of either I or II(See: FIG. 1A for structure and Examples 1 & 2 for preparation) inice-cold 0.5M MOPS buffer (pH 7.8 with NaOH) for 30 minutes on ice. Thereaction was terminated by the addition of TFA to 0.5% v/v finalconcentration. The modified peptides were then mixed in variouspre-determined proportions to approximately cover the range 1:10 to 10:1of the differentially labeled peptides. Each peptide mixture wasindividually purified by reverse-phase de-salting using a Millipore C18Zip-Tip. Excess reagent and buffer do not retain on the reverse-phasepackings, and were thus efficiently removed prior to MS analysis. Themixtures (0.5 μl) were then spotted onto a MALDI target plate,over-spotted with 0.5 μl of 1% w/v α-cyano cinnamic acid in 50% aqueousacetonitrile and each sample was analyzed using a MALDI source fitted toa QTOF analyzer.

FIG. 2 is an expansion plot of the MS spectrum obtained from the 1:1 mixof Glu-fibrinopeptide as modified with reagents I and II. The peak atm/z 1699 represents the N-terminally modified mass ofGlu-fibrinopeptide, and as expected, there is no observable differencein m/z of the two different forms of the peptide (See: FIGS. 1A(III) and1A(IV). The modified peptides are isobaric. The isotopic clusterobserved for the peak is exactly as expected for a single species.

The singly-charged precursor ion of m/z 1699 was then selected forfragmentation by low energy CID (collision offset of approximately−70V), yielding the MS/MS spectrum found in FIG. 3. As expected, theobserved ion series was predominantly of types b- and y-. All these ionsappeared as single species, with no indication that they comprised a 1:1mixture of the differentially-labeled peptide species. For example, anexpansion of the prominent y-ion at m/z 1056.5 is shown in the expansionplot as FIG. 4 and the prominent b-ion at m/z 886.3 is shown in theexpansion plot as FIG. 5. TABLE 1 Observed Predicted 0.13 0.125 0.170.166 0.2 0.25 0.46 0.5 1.03 1 2.15 2 4.16 4 6.3 6 7.9 8

Close examination of the spectrum at about 100 m/z (FIG. 6), however,reveals the presence of both species VII and VIII (FIG. 1B), which arethe fragmentation products of species V and VI (FIG. 1B), respectively.No peaks are observed at m/z 128.1, thereby indicating that species Vand VI are not stable enough to be observed. In this example, therefore,it may be that fragmentation of the amide bond between the carbonylgroup and the amino-terminal amino acid of the peptide (e.g. bond Y)induced subsequent fragmentation of the reporter/linker moiety (bond X)and loss of the carbonyl moiety as neutral CO. Peak integration wasperformed using the instrumentation provided with the instrument.Following compensation for the naturally occurring second C-13 isotopiccontribution of approximately 6 percent, the measured relative ratio ofVIII/VII (101/100) was 1.03 (expected value 1.00). Table 1 shows actualversus observed ratios for additional experimental mixtures prepared(ratio expressed as intensity m/z 101/m/z 100), with correction for thenaturally occurring second C-13 contribution. This data is alsorepresented graphically in FIG. 7. There is excellent agreement betweenobserved and predicted values, with mean error <10%.

Example 4 Proteomic Analysis

In practice, a representative proteomic analysis can be performed asfollows. Total cellular protein extracts for comparison (e.g. samples Aand B) are separately digested with trypsin, or another proteolyticenzyme. The resulting peptide mixtures are separately reacted withdifferent isomeric of isobaric labeling reagents (for example, compoundsI and II) to give complete modification of N-terminal and lysine aminesof the peptides. For example, sample A can be reacted with compound Iand sample B can be reacted with compound II. Each of the samplescontaining modified peptides/proteins are then be mixed together beforechromatographic separation (often using multi-dimensional HPLC) andanalyzed by MS and MS/MS techniques. The labeling can be performed witha single label treatment (no prior blocking of lysine groups with asecond reagent required) as the groups are isobaric.

The mixture of labeled proteins/peptides is then chromatographicallyseparated and the eluent, or fractions thereof, analyzed by massspectrometry as described in Example 3, above. Effective sensitivity mayalso be significantly increased using triple-quadruople or Q-trap massspectrometers, where the m/z region of 100 and 101 is monitored inprecursor-ion mode. The relative ratios of the two “signature” peaks aredirectly correlated with the ratio of each peptide/protein analyte ofinterest in each of samples A and B. As used herein, the “signature”peaks are the peaks for the reporter.

Example 5 Analyte Determination and Quantitation Relative to an InternalStandard

Total cellular protein extracts for comparison (e.g. samples A and B)are separately digested with trypsin. The resulting peptide mixtures areseparately reacted with X and XI (FIG. 8) to give substantially completemodification of N-terminal and lysine amines as described above. Forexample, sample A peptides are reacted with X and sample B peptides arereacted with XI. Known amounts or each of samples A and B, containingsubstantially modified peptides, are then mixed together. To thecombined mixture of A and B is now added, in accurately determinedamount, a set (one or more) of synthetic peptide(s) that correspondexactly in amino acid sequence and/or post-translational modification(e.g. phosphorylation) to peptide(s) that may be present in the mixtureof samples A and B, and where the synthetic peptide(s) are labeled withanother member of set of isobaric labeling reagents (e.g. compounds XIIor Xm, see: FIG. 8). The combined mixture of peptides from sample A,sample B and synthetic internal standard peptides can optionally besubjected to chromatographic separation, for example bymulti-dimensional HPLC, or electrophoretic separation and then analyzedby MS and MS/MS techniques as described previously. All equivalentlabeled peptides from sample A, B and synthetic counterparts ofidentical sequence are isobaric and have substantially identicalchromatographic properties. By “substantially identical chromatographicproperties” we mean that there is very little, if any, separation of thedifferentially labeled but otherwise identical peptides. Following MS/MSanalysis, the absolute concentration of peptides from sample A and B maybe accurately determined by comparison of the relative intensity of thereporters for X (sample A) and for XI (sample B) with respect to theintensity of the reporter (the “signature peak”) resulting from thestandard peptide labeled with the additional member of the isobaric set(e.g. XII or XIII).

Although the foregoing is a description of two samples (i.e. Samples Aand B), this process could be extended in many practical ways. Forexample, there may be many samples that are analyzed simultaneouslyprovided there is a large enough set of labeling reagents.

There could be a double (or more where there are more samples to beanalyzed) internal standard (e.g. sample A peptides may be ‘spiked’ withsynthetic peptides labeled with reagent XII and sample B peptides may bespiked with synthetic peptides labeled with reagent Xm (of knownabsolute concentration)). When all are combined, separated and analyzedas described above, Sample A peptides can be quantitated relative to thesignature peak for compound XII and sample B peptides can be quantitatedrelative to the signature peak for compound XIII.

Example 6 Exemplary Synthesis of Piperidine Acetic AcidN-hydroxysuccinimide Ester

Bromoacetic acid is dissolved in tetrahydrofuran (or another suitablenon-nucleophilic solvent) and added dropwise to a stirred solutioncontaining an excess of piperidine in tetrahydrofuran (THF, or anothersuitable non-nucleophilic solvent). The solution is stirred at roomtemperature for one to three days. The solid is filtered, washed withTHF (or another suitable non-nucleophilic solvent), and optionallyrecrystallised. For the two different isobaric versions of piperidineacetic acid, either bromoacetic-1-¹³C acid (Aldrich PN 27,933-1) orbromoacetic-2-¹³C acid (Aldrich PN 27,935-8) can be substituted forbromoacetic acid. Isomer substituted piperidine can be prepared fromsuitable starting material or it can be obtained, on a custom orderbasis, from sources such as Cambridge Isotope Laboratories or Isotec.

To convert the acetic acid derivatives to active esters, such as anN-hydroxysuccinimidyl ester, dimethylformamide (DMF) is dissolved intetrahydrofuran (or another suitable non-nucleophilic solvent). Thissolution is added dropwise to a stirred solution of an equal molaramount of thionyl chloride (based upon the molar quantity of DMF)dissolved in tetrahydrofuran (or another suitable non-nucleophilicsolvent) and cooled in an ice bath. After complete addition and 30minutes on ice, the ice bath is removed and solid N-hydroxysuccinimideis added immediately followed by piperidine acetic acid (or −1-¹³C or−2-¹³C piperidine acetic acid). The reaction is left vigorously stirringover night at room temperature. The product piperidine acetic acidN-hydroxysuccinimide ester is then isolated from the reaction mixturepossibly by mere filtration. Recrystallization and/or chromatography canoptionally be used to purify the crude product.

Example 7 Exemplary Synthesis of Piperazine Acetic AcidN-hydroxysuccinimide Ester

A solution containing two equivalents of piperazine dissolved intetrahydrofuran (THF) is added dropwise to a solution containing oneequivalent of bromoacetic acid (as compared with the amount ofpiperazine) dissolved in tetrahydrofuran. The two solutions should be asconcentrated as is practical. The resulting reaction solution is stirredat room temperature for one to three days. The solid is filtered, washedwith THF, and optionally recrystallised. For the two different isobaricversions of piperidine acetic acid, either bromoacetic-1-¹³C acid(Aldrich PN 27,933-1) or bromoacetic-2-¹³C acid (Aldrich PN 27,935-8)can be substituted for bromoacetic acid.

To convert the acetic acid derivatives to active esters, such as anN-hydroxysuccinimidyl ester, dry dimethylformamide (DMF, 1.75 g, 0.024M)can be dissolved in tetrahydrofuran. This solution can be added dropwiseto a stirred solution of an equal molar amount of thionyl chloride(based upon the molar quantity of DMF) dissolved in tetrahydrofuran andcooled in an ice bath. After complete addition and 30 minutes on ice,the ice bath can be removed and solid N-hydroxysuccinimide addedimmediately followed by piperazine acetic acid (or −1-¹³C or −2-¹³Cpiperidine acetic acid). The reaction can be left vigorously stirringover night at room temperature. The product piperazine acetic acidN-hydroxysuccinimide ester can then be isolated from the reactionmixture possibly by mere filtration. Recrystallization or chromatographycan then be used to purify the crude product.

Example 8 Exemplary Synthesis of N,N′-(2-methoxyethyl)-glycine ActiveEster (Copied From U.S. Pat. No. 6,326,479

To 1.1 mole of bis(2-methoxyethyl)amine (Aldrich Chemical) was addeddropwise 500 mmol of tert-butyl chloroacetate (Aldrich Chemical). Thereaction was allowed to stir for three days and was then worked up. Tothe final reaction contents was added 250 mL of dichloromethane (DCM)and 200 mL of water. To this stirring solution was added portionwise,300 mmol of solid potassium carbonate (K₂CO₃). After complete mixing,the layers were separated. The DCM layer was washed once with a volumeof water, dried (Na₂SO₄), filtered and evaporated to yield 66.3 g of avery thin yellow oil. This crude product was Kugelrohr distilled at 60°C. (200-500 μM Hg) to yield 58.9 g of a clear colorless oil (238 mmol;95%).

To the purified (stirring) N,N′-(2-methoxyethyl)-glycine-tert-butylester was slowly added 12.1 mL of concentrated hydrochloric acid. Thereaction was allowed to stir overnight and then the byproducts (e.g.water, HCl, isobutylene) were removed by vacuum evaporation. ¹H-MNRanalysis indicated the t-butyl ester was hydrolyzed but it appeared thatthere was water and HCl still present. The crude product wasco-evaporated 2×from acetonitrile (ACN) but water and HCl were stillpresent. To eliminate impurities, a 4.4 g aliquot was removed from thecrude product and Kugelrohr distilled at 135-155° C. (100-200 μM Hg withrapidly dropping pressure after distillation began). Yield 4.2 g (18.4mmol; 95% recovery of thick, clear, colorless oil). The distilledproduct did not contain any water or HCl.

The active ester (e.g. N-hydroxysuccinimidyl ester) of any suitableisotopically labelled substituted or unsubstitutedN,N′-(2-methoxyethyl)-glycine can then be prepared by methods known inthe art, such as those described herein.

Example 9 Exemplary Method for Preparing a Solid Support ComprisingLabelling/Tagging Reagents

A commercially available peptide synthesis resin comprising a“sterically hindered cleavable linker” is reacted with at least two-foldexcess of an aminoalkyl piperazine (e.g. 1-(2-aminoethyl)piperazine,Aldrich P/N A5,520-9; isomeric versions can be made by the processillustrated in FIG. 11 in combination with the description in thespecification). By “sterically hindered cleavable linker” we mean thatthe linker comprises a secondary or tertiary atom that forms thecovalent cleavable bond between the solid support and the atom or groupreacted with the cleavable linker. Non-limiting examples of stericallyhindered solid supports include: Trityl chloride resin (trityl-Cl,Novabiochem, P/N 01-64-0074), 2-Chlorotrityl chloride resin(Novabiochem, P/N 01-64-0021), DHPP (Bachem, P/N Q-1755), MBHA (AppliedBiosystems P/N 400377), 4-methyltrityl chloride resin (Novabiochem, P/N01-64-0075), 4-methoxytrityl chloride resin (Novabiochem, P/N01-64-0076), Hydroxy-(2-chorophnyl)methyl-PS (Novabiochem, P/N01-64-0345), Rink Acid Resin (Novabiochem P/Ns 01-64-0380, 01-64-0202),NovaSyn TGT alcohol resin (Novabiochem, P/N 01-64-0074). Excess reagentsare then removed by washing the support. The secondary amine of thesupport bound piperazine is then reacted with an excess of bromoaceticacid in the presence of a tertiary amine such as triethylamine. Excessreagents are then removed by washing the support. Depending on themethod to be used to make an active ester of the carboxylic acid (e.g.whether or not a salt of the carboxylic acid is required for the activeester synthesis), the wash can be selected to have a pH that is adjustedto protonate the support bound carboxylic acid group of thebis-alkylated piperazine. The carboxylic acid group of the support boundpiperazine is then converted to an active ester (e.g.N-hydroxysuccinimidyl ester) using procedures known in the art for theproduction of acid esters of a carboxylic acid, such as those describedabove. The resulting solid support can thereafter be used to labelanalytes of a sample (e.g. peptides) having nucleophilic functionalgroups. The labeled analytes can then be released from the support asdescribed by the manufacturer's product instructions. The product ofeach cleavage reaction can then be combined to form a sample mixture.

1. An active ester compound that is a 5, 6 or 7 membered heterocyclicring comprising a ring nitrogen atom that is N-alkylated with asubstituted or unsubstituted acetic acid moiety to which the alcoholmoiety of the active ester is linked through the carbonyl carbon of theN-alkyl acetic acid moiety, wherein the compound is isotopicallyenriched with one or more heavy atom isotopes.
 2. The compound of claim1, wherein the compound is isotopically enriched with three or moreheavy atom isotopes.
 3. The compound of claim 1, wherein theheterocyclic ring is substituted with one or more substituents.
 4. Thecompound of claim 3, wherein the one or more substituents are alkyl,alkoxy or aryl groups.
 5. The compound of claim 4, wherein the one ormore substituents are protected or unprotected amine groups, hydroxylgroups or thiol groups.
 6. The compound of claim 1, wherein theheterocyclic ring is aliphatic.
 7. The compound of claim 1, wherein theheterocyclic ring is aromatic.
 8. The compound of claim 1, wherein theheterocyclic ring comprises one or more additional nitrogen, oxygen orsulfur atoms.
 9. The compound of claim 1, wherein active ester is anN-hydroxysuccinimide ester.
 10. The compound of claim 1, wherein thecompound is a salt.
 11. The compound of claim 1, wherein the compound isa mono-TFA salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt. 12.The compound of claim 1, wherein each incorporated heavy atom isotope ispresent in at least 80 percent isotopic purity.
 13. The compound ofclaim 1, wherein each incorporated heavy atom isotope is present in atleast 93 percent isotopic purity.
 14. The compound of claim 1, whereineach incorporated heavy atom isotope is present in at least 96 percentisotopic purity.
 15. A kit comprising a compound as claimed in claim 1,further comprising one or more reagents, containers, enzymes, buffers orinstructions.
 16. The kit of claim 15, wherein the kit comprises aproteolytic enzyme.
 17. The kit of claim 15, wherein the kit comprisestwo or more compounds as claimed in claim 1, wherein said two or morecompounds are related as a set of isomers and/or isobaric labelingreagents.
 18. The kit of claim 17, wherein each labeling reagent isindependently linked to a solid support through a cleavable linker. 19.The kit of claim 15, wherein the compound has the formula:

wherein; a) RG is a reactive group that is an electrophile; b) Z is O,S, NH or NR¹; c) each J is the same or different and is selected fromthe group consisting of: H, deuterium (D), R¹, OR¹, SR¹, NHR¹, N(R¹)₂,fluorine, chlorine, bromine and iodine; d) W is an atom or group that islocated ortho, meta or para to the ring nitrogen and is selected fromthe group consisting of: NH, N—R², P—R², O or S; and e) each carbon ofthe heterocyclic ring has the formula CJ₂; f) each R¹ is the same ordifferent and is an alkyl group comprising one to eight carbon atomswhich may optionally contain a heteroatom or a substituted orunsubstituted aryl group wherein the carbon atoms of the alkyl and arylgroups independently comprise linked hydrogen deuterium and/or fluorineatoms; and g) R² is an amino alkyl, hydroxy alkyl, thio alkyl group or acleavable linker that cleavably links the reagent to a solid supportwherein the amino alkyl, hydroxy alkyl or thio alkyl group comprises oneto eight carbon atoms, which may optionally contain a heteroatom or asubstituted or unsubstituted aryl group, and wherein the carbon atoms ofthe alkyl and aryl groups independently comprise linked hydrogen,deuterium and/or fluorine atoms.
 20. A method comprising: a) reacting ananalyte with the compound as claimed in claim 1 to thereby produce alabeled analyte; and b) mixing the labeled analyte with one or moredifferentially labeled analytes.
 21. The method of claim 20, furthercomprising analyzing the mixture in a mass spectrometer.
 22. The methodof claim 20, wherein the analysis in a mass spectrometer involves MS/MSanalysis.